CD19-Targeted CAR-T Cell Therapy Research Articles

The Kinetics of Inflammation-Related Proteins and Cytokines in Children Undergoing CAR-T Cell Therapy-Are They Biomarkers of Therapy-Related Toxicities?

CD19-targeted CAR-T cell therapy has revolutionized the treatment of relapsed/refractory (r/r) pre-B acute lymphoblastic leukemia (ALL). However, it can be associated with acute toxicities related to immune activation, particularly cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Cytokines released from activated immune cells play a key role in their pathophysiology. This study was a prospective analysis of proinflammatory proteins and cytokines in children treated with tisagenlecleucel. Serial measurements of C-reactive protein, fibrinogen, ferritin, IL-6, IL-8, IL-10, IFNγ, and TNFα were taken before treatment and on consecutive days after infusion. The incidence of CRS was 77.8%, and the incidence of ICANS was 11.1%. No CRS of grade ≥ 3 was observed. All complications occurred within 14 days following infusion. Higher biomarker concentrations were found in children with CRS grade ≥ 2. Their levels were correlated with disease burden and CAR-T cell dose. While cytokine release syndrome was common, most cases were mild, primarily due to low disease burden before lymphodepleting chemotherapy (LDC). ICANS occurred less frequently but exhibited various clinical courses. None of the toxicities were fatal. All of the analyzed biomarkers rose within 14 days after CAR-T infusion, with most reaching their maximum around the third day following the procedure.

Abstract CT244: HR001, a novel CD19-targeted CAR-T cell therapy for patients with relapsed/refractory non-Hodgkin's lymphoma: primary results of a phase II study (HRAIN01-NHL01-II)

Abstract Background: HR001 is a CD19-targeted CAR-T cell for patients with relapsed and refractory non-Hodgkin's (R/R NHL). Here, we report the primary results of the phase 2 study (HRAIN01-NHL01-II). Methods: HRAIN01-NHL01-II is an open-label, single-arm, and multicenter (17 centers) study in China. It enrolled R/R NHL patients treated with at least two lines of therapy (must-have anthracycline and CD20-targeted agents) or after autologous hematopoietic stem cell transplantation. All patients received a single infusion of HR001 at the 2.0 × 106 CAR+T cells/kg dose. The disease response was evaluated by the independent review committee with Lugano response criteria [2014]. Results: As of May 16th, 2023, 81 R/R NHL patients with ECOG 0-1 at baseline received the infusion HR001 from the Jinshan manufacturing facility [51.9% male; median age 53.4 years (range 23~74); 3.7% CD19 negative; 91.4% diffuse large B-cell lymphoma; 79.0% III-IV of Ann Arbor classification or Lugano] with a median follow-up of 160 days (range 7 to 454). The median time to best response was 30 days (range 28-358). The 3-month, 6 month and best objective response rate (ORR) was 53.1%(95% CI: 41.7-64.3), 45.7%(95% CI: 34.6-57.1) and 74.1% (95% CI: 63.1-83.2) respectively. The 3-month, 6 month and best complete response rate (CRR) was 32.1%(95% CI: 22.2-43.4), 29.6%(95% CI: 20.0-40.8) and 49.4%(95% CI: 38.1-60.7) respectively. Median duration of response (DOR) and progression-free survival (PFS) were 339 days (95% CI: 149-NE) and 176 days (95% CI: 91-NE) respectively. The overall survival (OS) was not reached. The most common treatment-related adverse events (AEs) happened in the hematologic system. The cytokine release syndrome (CRS) occurred in 95.1% of patients, with ≥3 grade CRS observed in 3.7% of patients. Immune effector cell-associated neurotoxicity syndrome (ICANS) happened in 8.6% of patients, without any ≥ 3 grade ICANS reported. The treatment-related mortality was 1.2%. The median time to CAR T-cell peak expansion was 240 hours (120~672) after infusion, median maximum expansion (Cmax) was 17413.9 copies/μg DNA (124.9~136382.2), and median AUC0-28d was 3201014.0 copies/μg DNA (205082.8~18400016.2). Positive tests for the anti-drug antibody (ADA) were noted in 14.8% of patients, while neutralizing antibody (Nab) tests were positive in 3.7% of patients. Among Nab-positive patients, the complete response was maintained until follow-up. Conclusions: Patients with R/R NHL could benefit from the HR001 treatment with satisfactory objective response, durable remission, and favorable safety profile. Citation Format: Yian Zhang, Yuhua Li, Kai Hu, Wuping Li, Hongwei Xue, Qi Deng, Zheng Ge, Liangquan Geng, Xianmin Song, Kang Yu, Hongmei Jing, Bing Chen, Xin Li, Zhimin Zhai, Jianping Shen, Jiang Cao, Qingming Wang, Peng Liu. HR001, a novel CD19-targeted CAR-T cell therapy for patients with relapsed/refractory non-Hodgkin's lymphoma: primary results of a phase II study (HRAIN01-NHL01-II) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT244.

T-Cell Large Granular Lymphocyte Population Involving Chimeric Antigen Receptor-Modified T (CAR T) Cells in Patients with Cytopenia after CD19-Targeted CAR T-Cell Therapy: Case Series

T-Cell Large Granular Lymphocyte Population Involving Chimeric Antigen Receptor-Modified T (CAR T) Cells in Patients with Cytopenia after CD19-Targeted CAR T-Cell Therapy: Case Series

Low-Dose Targeted Radioimmunotherapy (Iomab-ACT) Achieves Lymphocyte and Monocyte Depletion Prior to CD19-Targeted CAR T-Cell Therapy for Relapsed of Refractory B-Cell ALL or DLBCL with Minimal CRS and Icans

Low-Dose Targeted Radioimmunotherapy (Iomab-ACT) Achieves Lymphocyte and Monocyte Depletion Prior to CD19-Targeted CAR T-Cell Therapy for Relapsed of Refractory B-Cell ALL or DLBCL with Minimal CRS and Icans

Low-Frequency PPM1D Gene Mutations Affect Treatment Response to CD19-Targeted CAR T-Cell Therapy in Large B-Cell Lymphoma.

Chimeric antigen receptor T (CAR T)-cell therapy has become a standard treatment option for patients with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Mutations in the PPM1D gene, a frequent driver alteration in clonal hematopoiesis (CH), lead to a gain of function of PPM1D/Wip1 phosphatase, impairing p53-dependent G1 checkpoint and promoting cell proliferation. The presence of PPM1D mutations has been correlated with reduced response to standard chemotherapy in lymphoma patients. In this study, we analyzed the impact of low-frequency PPM1D mutations on the safety and efficacy of CD19-targeted CAR T-cell therapy in a cohort of 85 r/r DLBCL patients. In this cohort, the prevalence of PPM1D gene mutations was 20% with a mean variant allele frequency (VAF) of 0.052 and a median VAF of 0.036. CAR T-induced cytokine release syndrome (CRS) and immune effector cell-associated neuro-toxicities (ICANS) occurred at similar frequencies in patients with and without PPM1D mutations. Clinical outcomes were globally worse in the PPM1D mutated (PPM1Dmut) vs. PPM1D wild type (PPM1Dwt) subset. While the prevalent treatment outcome within the PPM1Dwt subgroup was complete remission (56%), the majority of patients within the PPM1Dmut subgroup had only partial remission (60%). Median progression-free survival (PFS) was 3 vs. 12 months (p = 0.07) and median overall survival (OS) was 5 vs. 37 months (p = 0.004) for the PPM1Dmut and PPM1Dwt cohort, respectively. Our data suggest that the occurrence of PPM1D mutations in the context of CH may predict worse outcomes after CD19-targeted CAR T-cell therapy in patients with r/r DLBCL.

The Bengaluru Score - a Prognostic Predictive Score for Clinical Efficacy Outcomes Based on Discovery Cohort of Patients Treated with Varnimcabtagene Autoleucel (IMN-003A) in the Phase-2 (IMAGINE) Study

Background: Recurrence of disease is seen in some patients post CD19 targeted CAR-T cell therapy. There is a need to develop CAR-specific efficacy risk-classification systems as established parameters of disease relapse are not predictive. In the Phase-2 IMAGINE study, multiple variables that may predict clinical outcomes were studied and the relevant clinical, laboratory and translational information were integrated. This abstract reviews the development of the Bengaluru Score as a predictive prognostic score for clinical efficacy outcomes based on discovery cohort of patients treated with varnimcabtagene autoleucel (IMN-003A). Methods: Multiple characteristics for disease progression were reviewed (n=36) and risk stratified as: 1) Baseline parameters (age, lines of treatment, bridging therapy, cytogenetics, blasts, total metabolic tumour volume (TMTV), sum of perpendicular diameter (SPD), refractory, relapse within 6m, laboratory parameters - haemoglobin, neutrophil count, lymphocyte count, platelet count, CRP, LDH, ferritin, baseline interleukin-6 (IL-6), baseline total T cells, CD4, and CD8 subsets in peripheral blood); 2) Apheresis (total T cells, naïve / central memory in CD4 and CD8 subsets); 3) Final Product characteristics [total CAR+ cells, naïve / central memory in CD4 and CD8 subsets, CD8 (naïve & central memory subsets / tumour burden (TMTV and SPD), product doubling time] and 4) Post Infusion (CAR peak & persistence, B cell aplasia, hypogammaglobulinemia, infections and intravenous immunoglobulin (IVIg) replacement). These parameters were analyzed using chi-square and appropriate multivariant odds ratio (OR) to examine the association with disease progression. Independent risk factors were identified and weighted score allocated for OR ≥10 to obtain a multifactorial prediction model - The Bengaluru Score. The specificity and sensitivity of this model was then studied in the discovery cohort. Results: In the IMAGINE study, with ORR 80.9% at primary endpoint (n=17/21) and median PFS not reached (range 12 to NR), relapse was seen in 37.5% (n=9/24) pts with median PFS of 178 days (range 28 to 319). Multivariant analysis of these parameters showed some statistical significance for following variables: Baseline Platelet count <200 x 10 9/L (overall); Final Product doubling time ≥1 day (B-ALL); Baseline SPD ≥2000 mm 2, Hb <12 g/dL, Platelet count <200 x 10 9/L and post infusion IVIg replacement (B-NHL), Of these variables, statistically significant association was seen with Final Product doubling time ≥1 day (B-ALL) and Baseline SPD ≥2000 mm 2 with Hb <12 g/dL and Platelet count <200 x 10 9/L (B-NHL). The impact of IVIg replacement on efficacy needs further study. A multiple factor-based prognostic score system - The Bengaluru Score was developed classifying patients into B-ALL low-risk (1 point) and high-risk (3 points) and for B-NHL low risk (<5 points) and high-risk (≥5 points), Table 1. The Bengaluru Score when applied to discovery cohort in the IMAGINE study was able to risk stratify patients for disease progression with Sensitivity, Specificity, Positive Predictive Value and Accuracy of 100.00%, 66.67%, 60.00% and 77.78% respectively (for B-ALL) and 100.00%, 100.00%, 100.00% and 100.00% respectively (for B-NHL), Table 2. Conclusions: The Bengaluru Score is probably the first predictive prognostic score for clinical efficacy outcomes and is based on multivariant statistical analysis in the discovery cohort of patients in the IMAGINE study integrating clinical, laboratory and translational data with a very high sensitivity, specificity and accuracy. The score highlights the role of overall T cell expansion in the final product for B-ALL (but not the T N+CM subsets on its own) and for B-NHL, disease burden and haematological reserve. This suggests that earlier use of varnimcabtagene autoleucel may positively influence efficacy outcome but needs to be proven in the real-world setting. The Bengaluru Score can guide informed treatment decisions and evolve clinical strategies, such as reinfusion, CAR-T targeting a different antigen or allogeneic transplant (if applicable), to reduce the risk of relapse / disease progression in high-risk patients. The Bengaluru Score needs validation in a larger cohort of patients following treatment with varnimcabtagene autoleucel and other CD19 targeted CAR-T cell therapies.

Phosphorus Disruption Is Associated with the Incidence and Severity of Neurotoxicity Symptoms in CD19-Targeted CAR-T Cell Therapy: A Pooled Clinical Trial Analysis

Background Hypophosphatemia due to increased cell metabolic activity as seen in refeeding syndrome and sepsis, has a similar neurologic presentation to immune effector cell-associated neurotoxicity syndrome (ICANS), an associated risk of CAR-T cell therapy. CAR-T patients who develop ICANS are observed to have a higher incidence and more severe degrees of hypophosphatemia (Tang et al, 2022). We further explored the association of hypophosphatemia and other electrolyte disturbances with ICANS incidence across pooled CAR-T clinical trial patients to assess how serum electrolytes correlated with risk of ICANS. Methods We analyzed 593 patients with relapsed/refractory B-cell acute lymphoblastic leukemia or non Hodgkin's lymphoma, with ICANS (n=325), and without ICANS (n=268) treated with CD19-targeted CAR-T cell therapy across pooled clinical trial data from the Medidata Enterprise Data Store. Patients were grouped by ICANS status. A Fisher exact test was performed for categorical variables. A Mann-Whitney was performed for numerical variables. Variables of interest were baseline patients characteristics (age, gender, indication) and ICANS status. Common lab values investigated were nadir values and time to nadir for phosphate (Phos), magnesium (Mg), calcium (Ca), and potassium (K), peak value for C-reactive protein (CRP), and creatinine (Cr) at the time of infusion. To assign a hypo- phosphatemia, -kalemia, -magnesemia and -calcemia status to each patient, we used thresholds of <2 mg/dL, 3.5 mmol/L, 1.5 mg/dL and 2.0 mmol/L respectively. We performed a univariable logistic regression with ICANS status as the outcome variable and the following: Baseline value at infusion for Mg, K, Phos, Ca, CrNadir values between day 0 and 14 for Mg, K, Phos, Ca, CrSlope (between baseline and nadir) for Mg, K, Phos, Ca, CrPeak CRP, Cr and Urate between day 0 and 14 The odds ratios were calculated. Kaplan Meier curves were plotted. The log rank test was performed for time to event of ICANS, grouped by hypo- phosphatemia, -kalemia, -magnesemia and -calcemia status. We performed an analysis on the effect of the preemptive use of electrolyte replacement. We defined preemptive use as any electrolyte replacement given to patients either before the occurrence of ICANS or if patients did not get ICANS. For those with hypophosphatemia, we examined the preemptive use of electrolyte replacements for any electrolyte excluding Phos and any electrolyte with Phos. For those 2 groups we compared the rates of ICANS. Results CRS, and hypo- phosphatemia, -kalemia, -magnesemia and -calcemia status were statistically significantly different between ICANS and no ICANS groups. When looking at the nadir values, these 4 electrolyte disturbances were also statistically significant, with lower values associated with ICANS. For the time to nadir, only K was significant and higher for the ICANS group. Peak CRP was also higher for patients with ICANS, but did not meet the 0.05 threshold to be statistically significant. For the univariate logistic regression, variables positively associated with ICANS and significant were peak values for creatinine and CRP, OR of 3.8 (1.6-9.2) and 2.1 (0.9 - 5.0) respectively. Variables negatively associated with ICANS and significant were nadir values for Phos (OR 0.3 (0.1 - 0.8)), K (OR 0.2 (0.1-0.7)), Ca (OR 0.2 (0.1-0.6)), Mg (OR 0.17(0.1-0.5)) and urate (0.3(0.1-1)), and slope for Mg (OR 0.0 (0.0-0.2)). When plotting the Kaplan Meier curves, ICANS seems to develop sooner for patients with either hypo- phosphatemia, -kalemia, -magnesemia or -calcemia and the log rank test showed to be statistically significant (Figure 1). Among the patients with hypophosphatemia, the use of Phos as part of electrolyte replacement reduces the ICANS rate: 52% (0.39-0.65) versus 71% (0.58-0.84) for the electrolyte replacement without Phos. Conclusion Our data demonstrate that ICANS incidence is associated with disruptions in serum phosphorus levels, and that supplementation of phosphorus is able to mitigate the incidence of ICANS as well. Monitoring phosphorus may serve as a useful biomarker for ICANS, while goal directed phosphorus supplementation may serve as an inexpensive and readily available means to treat or prevent ICANS. Prospective studies with goal-directed phosphorus repletion are necessary to further study the therapeutic impact of electrolyte supplementation on ICANS incidence and severity.

Rituximab Ibrutinib Lenalidomide (R2I) Combination for Primary or Secondary Large B-Cell CNS Lymphoma: Last Resort or Bridge to Cellular Therapy?

Background: Primary central nervous system lymphoma (PCNSL) and secondary central nervous system lymphoma (SCNSL) each carry a poor prognosis. About 50% of patients with PCNSL will have refractory or relapsed disease correlating with dismal survival. SCNSL is associated with median overall survival (OS) of <6 months. (El-Galaly et al . Eur. J. Cancer 2018) Lenalidomide and ibrutinib each have single agent activity in PCNSL and SCNSL, albeit with limited duration of response (DOR). In combination, they have synergistic activity through modulation of NF-kappa B pathway. The regimen rituximab, lenalidomide, and ibrutinib (R2I) has demonstrated complete and durable responses in a subset of patients with good tolerability. (Houllier et al . Neurology 2021). We report our institutional outcomes with this regimen and its potential role as a bridge to autologous stem cell transplantation (ASCT) or CD19-targeted CAR T-cell therapy. Methods: This retrospective study included ten large B-cell lymphoma (LBCL) patients with PCNSL or SCNSL who received R2I regimen at Miami Cancer Institute from December 2020 to March 2023. Treatment response was assessed using International PCNSL Collaborative Group's criteria. (Abrey et al. J Clin Oncol 2005) Efficacy was evaluated from the start of R2I regimen. Progression free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Results: The median age at R2I initiation was 71 (range, 57-85) years. Six of 10 (60%) patients were ≥65 years. Five of 10 (50%) patients had PCNSL. The median number of prior lines of therapy was 2 (range, 1-7). None of the patients had prior ASCT. The median number of cycles of R2I was 4 (range, 1-23). Four patients received a higher dose of rituximab (500mg/m 2). Overall response rates (ORR) and complete response (CR) rates were 80% (95% CI, 55.2 to 100) and 40% (95% CI, 9.6 to 70.3), respectively. The median time to best response was 1.5 (range, 1.0-13.0) months and the median DOR was 5.5 (range, 2.5-18.0) months. The 1-year PFS and OS were 38.8% (95% CI: 4.9 to 72.8) and 56% (95% CI: 22.5 to 89.4), respectively (Figure 1). In the PCNSL subgroup, the 1-year OS was 80% (95% CI, 44.9 to 100) and PFS was 60% (95% CI, 17 to 100). No differences in response rates to R2I between PCNSL and SCNSL were observed (p=0.9). There were no deaths or treatment discontinuations due to R2I toxicity. Two patients on R2I underwent subsequent cellular therapy. One PCNSL patient in CR received carmustine (BCNU) and thiotepa conditioning followed by autograft rescue and remains in remission 16 months post-ASCT. The second patient received CD19-targeted CAR T-cell therapy with axicabtagene ciloleucel for SCNSL. The patient expired 119 days after CAR T-cell therapy due to systemic disease relapse. Another patient, initially deemed ineligible for any cellular therapy due to poor performance status, has achieved a durable response with improvement of performance status and is currently awaiting ASCT. Conclusion: R2I is an effective and safe regimen in patients with PCNSL and SCNSL. Although responses without consolidation may be durable, R2I should be considered as an active drug combination and well-tolerated bridge to cellular therapy to achieve long term survival.

Machine Learning-Based Decision Tree for Identifying and Grading Severity of Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) Based on Neurological Adverse Events in Patients Treated with CD19 CAR-T

Introduction One of the major challenges associated with Chimeric Antigen Receptor T-cell (CAR-T) therapy is the development of immune effector cell-associated neurotoxicity syndrome (ICANS), which can vary in severity and requires prompt intervention. This study proposes using machine learning in combination with clinical insight to build a decision tree that accurately identifies and grades ICANS severity in patients treated with CD19 CAR-T based on the neurological adverse events (NAEs). Methods We conducted a retrospective analysis of 384 patients that exhibited NAEs after being treated with CD19-targeted CART-cell therapy in the pooled clinical trial data from the Medidata Enterprise Data Store. Of note, this dataset excludes patients without NAEs; these patients were assumed to not experience clinical ICANS. Patient NAEs were reviewed and the highest ICANS grade was assigned by a neuroimmunologist, resulting in the following patient counts: grade 0 (n=61), 1 (n=69), 2 (n=166), 3 (n=75), 4 (n=13). The clinical expert graded each patient based on the NAEs recorded in the first 28 days post infusion during the clinical trial, their CTCAE toxicity and symptom onset day. To process NAEs, we first mapped each of the 219 verbatim terms to the MedDRA hierarchy using a proprietary Medidata coding algorithm. We then aggregated the NAEs to the High Level Group Term (HLGT) level except for the MedDRA term “Neurological Disorders NEC” which was broken down to the High Level Term (HLT) level due to the large size of this group (73 out of 219 terms).The mapping of NAEs to MedDRA groups was reviewed together with the neuroimmunologist. The final dataset included the CTCAE severity of the NAEs mapped to MedDRA groups as features and the expert-labeled ICANS grades as the target variable. The data was then split into train (70%) and test (30%) sets stratifying by study and ICANS grade. We fit a decision tree while performing 5-fold hyperparameter grid search on the train set. The tree with the highest area under the Receiver Operating Characteristic curve one-vs-one (ROCAUC OVO) was selected and evaluated on the test set using an array of classification performance metrics. The model output was evaluated at the ICANS grade and severity (no/moderate/severe) level. The confusion matrices for the test set and the full decision tree were plotted. Results When binning the ICANS grade prediction by severity (grade 0 = no ICANS, grade 1-2 = moderate, grade 3-4 = severe), the model achieves a strong out-of-sample accuracy of 83.6% and ROCAUC OVO of 0.935. Regarding the individual ICANS grade prediction, the model yielded lower out-of-sample accuracy of 54.3%, with the majority of errors between grades 1 and 2. In spite of this, there was still reasonable discrimination based on the out-of-sample ROCAUC OVO values of 0.863. The training algorithm selected six MedDRA groups of NAEs to make predictions with: Headaches, Seizures (incl subtypes), Encephalopathies, Cortical dysfunction NEC, Disturbances in consciousness NEC, and Movement disorders (incl parkinsonism). We observe that patients with only headaches (a separate feature in our dataset) are all labeled as having no ICANS while those with seizures of grade 2 and higher are classified as grade 4. Refer to Figure 1 for the full decision tree. Comparing the neurologist-assigned versus predicted ICANS grades in the out-of-sample patient group (Figure 2) revealed that the majority of errors were bound to +/- one grade. Only three out of 116 patients were mislabeled by two grades. Crucially, none of the grade 1+ patients were predicted by the model to have grade 0. Conclusion Our machine learning-based decision tree accurately predicts the severity and grade of ICANS in CD19 CAR-T treated patients, aiding in clinical decision-making as well as retrospective analyses of ICANS patients. This type of clinical decision support tool has the potential to be a powerful aid to physicians by providing them with earlier physiological insights and data-driven assessments to help them triage patients accordingly. The model's performance highlights its potential clinical utility in improving patient care and outcomes. Further validation across multiple neurologists and prospective studies are warranted to assess its generalizability and refine its predictive capabilities. Acknowledgments We thank the developers of Scikit-learn and Dtreeviz Python packages for open-sourcing their code.

Phenotypic, Transcriptomic and Proteomic Characteristics of CAR T-Cell Dysfunction Are Associated with Inferior CAR T-Cell Expansion and Treatment Failure in r/r B-NHL

Introduction: Despite the curative potential of CAR T-cells, a significant number of patients fail to respond or relapse early. Recently, we and others showed that early expansion failure, and particularly CAR T-cell levels at day 7, are strong predictors for treatment resistance (Blumenberg et al, Blood Supplement 2022; Locke et al, Blood Adv 2020). Here we dissected early characteristics of CAR T-cell dysfunction based on immune checkpoint expression, transcriptomic signatures and cytokine profiles and investigated their link to CAR T-cell expansion failure and patient outcome of CD19-targeted CAR T-cell therapy. Methods: Patients with r/r B-NHL who underwent treatment with axi-cel or tisa-cel between January 2019 and November 2021 in the third- or later-line setting at the LMU in Munich were included (n=55). EDTA-anticoagulated peripheral blood and serum was collected. CD19 CAR and immune checkpoint expression (IC; PD-1, TIM-3, LAG-3 and CD224) was assessed by multiparameter flow cytometry on day 14 after CAR T-cell infusion. A next-generation targeted single-cell proteogenomics approach (BD Rhapsody TM) to analyze the transcriptome of distinct CAR T-cell subpopulations on day 7 and day 28 after CAR T-cell infusion. A total of 92 human immuno-oncology related proteins were simultaneously measured using the Olink® Immuno-Oncology panel on day 14 after infusion. Signatures of CAR T-cell dysfunction have been compared between patients with low (loEx) or high (hiEx; < vs ≥ 19 CAR T-cells/µl at day 7 post infusion)CAR T-cell expansion and were put into context of clinical outcome measures. Results: CAR T-cell levels at day 7 were inversely correlated to co-expression of PD-1 (r = -0.4923, p = 0.0011, n = 41), TIM-3 (r = -0.3634, p = 0.0195, n = 41) and LAG-3 (r = -0.5412, p = 0.0003, n = 41) on CAR T-cells at day 14, suggestive of an association of early expansion failure and dysfunction of CAR T-cells. Indeed, loEx (n=26) showed higher frequencies of CAR T-cells coexpressing several IC, including LAG-3 + (p=0.0036), LAG-3 +PD-1 + (p = 0.0048), or LAG-3 +TIM-3 + (p = 0.0065) compared to hiEx (n=15) on day 14 after transfusion. Conversely, we detected higher frequencies of non-dysfunctional CAR T-cell phenotypes negative for several IC, such as CD244 +PD-1 -TIM-3 - (p = 0.0006), CD244 +LAG-3 -PD-1 -TIM-3 - (p = 0.001) and CD244 +LAG-3 +PD-1 -TIM-3 - (p = 0.011) in hiEx. Next, we applied uniform manifold approximation and projection (UMAP) data to examine the surface expression of IC on CAR T-cells at day 14 post-infusion according to treatment response. Unsupervised clustering revealed a higher abundance of CAR T-cell clusters positive for several IC (CD244 +LAG-3 -PD-1 +TIM-3 +) in NR (n = 16) compared to R (n = 23). By contrast, CAR T-cell clusters containing CAR T-cells expressing no IC were more abundant in R compared to NR. In addition, we found significantly increased levels of a highly dysfunctional CAR T-cell phenotype positive for all four IC in NR compared to R at day 14 post infusion (p = 0.0178, n = 46). When specifically comparing the transcriptome of CAR T-cells between NR (n = 6) and R (n = 6), immune inhibitory and exhaustion-related genes were upregulated in NR, such as GZMK, KLRC1 and NR4A2 (figure 1, cluster 1). Conversely, CAR T-cells in R overexpressed genes related to T-cell effector function and cell differentiation, such as TIMP-1, TNFDF10 and MYC (figure 1, cluster 0). Consistent with these findings, a multiplexed proteomics assay for immunomodulatory serum factors in day 14 samples revealed an upregulation of proteins linked to an immune inhibitory and inflamed milieu in patients with highly dysfunctional CAR T-cells (e.g., sPD-L1 and sLAG-3, p = 0.0235 and p = 0.0264, respectively, n = 46) as well as in non-responding patients (e.g., sPD-L1 and IL-6, p = 0.0036 and p = 0.0053, respectively, n = 55). Conclusion: We were able to characterize a phenotypic, transcriptomic and proteomic signature of CAR T-cell dysfunction in post-infusion samples of both patients experiencing CAR T-cell expansion failure as well as lack of radiographic response. Our data show that treatment failure might be predicted not only by CAR T-cell kinetics but also by early assessment of the CAR T-cell phenotype, transcriptome and secretome, and thus enables identification of patients in need of salvage treatment.

Phase 2 Study of CD19 CAR T Cells with a Fully Human Binder for Large B-Cell Lymphoma in Relapse or Progression after a Murine Binder-Bearing CD19 CAR T-Cell Therapy

BACKGROUND T cells engineered with a CD19-targeted chimeric antigen receptor (CD19 CAR T cells) have revolutionized the treatment of high-risk, relapsed, or refractory large B-cell lymphoma (LBCL), but durable responses are only achieved in 30-40% of patients (pts). Outcomes are dismal in case of disease recurrence after CD19 CAR T-therapy; in this situation we have previously reported low efficacy and limited duration of response after a second infusion of the same CD19 CAR T-cell product (Gauthier, Blood, 2022). We observed that repeat CD19 CAR T-cell infusions were impaired by CD8+ T-cell responses against murine amino acid sequences located in the single chain variable fragment (scFv) of the CAR, which led to rapid immune rejection of CAR T cells. We hypothesized that CAR T cells engineered with a fully human scFv could circumvent anti-murine scFv immune responses in pts previously exposed to murine scFv-bearing CAR T cells. We report our phase 2 clinical trial results investigating treatment with fully human scFv-bearing CD19 CAR T cells (JCAR021) in R/R LBCL pts with relapsed or progressive disease after murine scFv-bearing CD19 CAR T-cell therapy. METHODS Pts with R/R LBCL were enrolled at the Fred Hutchinson Cancer Center on a phase II study (NCT03103971). Key eligibility criteria included: i) relapsed disease after complete response (CR) to prior CD19-targeted non-JCAR021 CAR T-cell therapy or persistent disease after partial response to prior CD19-targeted non-JCAR021 CAR T-cell therapy; ii) CD19-positive disease since completing the prior CD19-targeted CAR T-cell therapy. Pts received lymphodepletion (LD) with cyclophosphamide 300 mg/m2/d and fludarabine 30 mg/m2/d for 3 days. Cytokine release syndrome (CRS) and neurotoxicity (NT) were graded according to the Lee 2014 criteria and CTCAE v4.03 criteria, respectively. Disease response was evaluated at day +28 after JCAR021 infusion per PET-CT Lugano criteria. The planned sample size and CAR T-cell dose were n=27 and 7x10 6 CAR T cells/kg, respectively. RESULTS Pt characteristics, and outcomes are shown in the Table. All eight screened pts were enrolled, received LD, and JCAR021 infusion. Median age at enrollment was 63 (range: 41-77). Prior to lymphodepletion, 4 pts (50%) had bulky disease (largest lesion ≥5cm). All pts had extranodal disease. We observed elevated LDH prior to lymphodepletion in 6 pts (75%). Median prior lines of therapy was 5 (range: 3-7). Prior CD19 CAR T-cell product type was lisocabtagene maraleucel, n=4 (50%); axicabtagene ciloleucel, n= 3 (37%); or tisagenlecleucel, n=1. Response to the prior CD19 CAR T-cell therapy was CR in 5 pts (62%) and PR in 3 pts (38%). Median time from the first CAR T-cell infusion to relapse or progression was 165 days (range 49-434). Median time from the first CAR T-cell infusion to JCAR021 infusion was 304 days (range: 202-603). JCAR021 was administered at the dose of 7x10 6 (n=6, 75%) or 2x10 6 CAR T-cells/kg (n=2, 25%; dose reduction due to grade 5 NT in subject #6). We observed all-grade CRS in 4 pts (50%; grade 1, n=2; grade 2, n=2 not requiring vasopressors) and NT in 1 patient (dose limiting toxicity, grade 5 NT from intracerebral hemorrhage with concurrent acute kidney injury and toxic encephalopathy). At day+28 responses were seen in 3 of 8 pts (37%; CR, n=2; PR, n=1), all of whom received 7x10 6 CAR T-cells/kg. Two pts in CR after JCAR021 had bulky disease and elevated LDH prior to LD. In both pts, the prior CD19 CAR T-cell therapy had induced CR lasting ≥ 6 months. In the two evaluable responders (death in CR, n=1), we observed disease relapse or progression within 3 months. Median overall survival was 105 days (95%CI, 92-NA). We measured expansion of JCAR021 CAR T-cells in all pts. Exploratory analyses showed lower in vitro CAR T-cell expansion during manufacturing (fold expansion of CD8+ CAR T-cells, p<0.001) and lower in vivo CAR T-cell expansion (peak CD8+ CAR-T, p=0.002; AUC day0-28, p=0.01; JCAR021 dose, 7x10 6/kg) compared to pts receiving JCAR021 as their first CD19 CAR-T product (CAR-naïve cohort). CONCLUSION In LBCL pts with disease relapse or progression after a first CD19 CAR T-cell therapy, we observed low response rates and lack of durable responses after treatment with the fully human scFv-bearing product JCAR021, prompting early study termination. Lower CAR T-cell expansion during manufacturing in CAR-exposed pts suggest pre-existing T-cell dysfunction as a potential mechanism of failure.

Poor Hematopoietic Reserve at the Time of CD19 CAR T-Cell Infusion Is Associated with Long Term B-Cell Aplasia

Background: On-target off-tumor toxicities of anti-CD19 CAR T-cell therapy include B-cell aplasia and hypogammaglobulinemia. Long term B-cell aplasia is hypothesized to represent functional CAR T-cell engraftment (Melenhorst, Nature 2022), while peripheral blood B-cell recovery represents the loss of functional CAR-T cells against target over time. Patients with large B-cell lymphoma (LBCL) who are clinical responders have a 50-70% chance of B-cell recovery in the first year, with the remainder experiencing longer term B-cell aplasia (Logue, Haematologica 2021). Here, we sought to identify factors associated with long term B-cell aplasia using a cohort of responding patients who received CAR T-cell therapy for LBCL. Methods:This retrospective cohort study included 57 patients with LBCL treated with CD19-targeted CAR T-cell therapy between June 2016 and August 2020 at Moffitt Cancer Center who exhibited complete or partial response at six months after CAR T-cell infusion. Patients were treated with with axicabtagene ciloleucel (axi-cel; n=50) or tisagenlecleucel (tisa-cel; n=7), either as standard-of-care therapy (n=47) or as part of a previously published clinical trial (n=10; NCT02348216, NCT03391466, NCT03153462). Peripheral blood B-cells were measured by flow cytometry using CD19 expression as part of routine clinical care at baseline and periodically after infusion. Prolonged B-cell aplasia was defined as a CD19+ B cell count comprising less than 1% of peripheral blood mononuclear cells during a minimum of two separate time points beyond 6 months of follow-up. Patients were defined as having B cell recovery if >1% B-cells were observed in peripheral blood at any time after CAR T-cell infusion. Results: At a median follow up of 33 months (95% CI: 28.7, 37.3 months), 29 (51%) patients had persistent B-cell aplasia and 28 (49%) had recovery of peripheral B-cell counts. Median B-cell count at 12 months in the recovery group was 84/µL (IQR 2.5-197) compared to 0/µL (IQR 0-1) in the aplasia group. Patients with long-term B-cell aplasia had lower CD4 T-cell counts prior to CAR T-cell infusion [median 200/µL (IQR 136-278) vs. 330/µL (IQR 224-451); P= 0.008] and over time compared to patients with B-cell recovery (Fig. A). Patients with long-term B cell aplasia also had lower pre-infusion absolute neutrophil counts (2.3K/µL (IQR 1.4-4.15) vs. 3.5K/µL (IQR 2.8-4.2); P<0.001) and higher baseline CAR-HEMATOTOX scores [2 (IQR 1-3.25) vs 1 (IQR 0-1)], consistent with poor baseline hematopoietic reserve (Rejeski, Blood 2021). While disease-related mortality did not significantly differ between groups, non-relapse mortality was markedly higher in patients with long-term B-cell aplasia due to infection: all 8 patient deaths in the aplasia group were due to infections occurring beyond day 30 post-infusion, with 6 of the 8 deaths due to COVID-19. However, no patients in the B-cell recovery group died of late infection (Fig. B). Conclusions: Long term B-cell aplasia is hypothesized to represent ongoing CAR T-cell activity against normal B-cells. However, our results demonstrate that B-cell aplasia is associated with low baseline hematopoietic reserve and subsequent poor immune reconstitution of multiple lineages including B-cells, T-cells, and myeloid cells. Further studies are warranted to assess whether poor immune reconstitution and B-cell aplasia are related to long term CAR T-cell activity or other processes. These patients are at higher risk for non-relapse mortality from infection, and mitigation strategies are needed.

Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations

Constant challenges for the treatment of mantle cell lymphoma (MCL) remain to be recurrent relapses and therapy resistance, especially in patients harboring somatic mutations in the tumor suppressors ATM and TP53, which are accumulated as therapy resistance emerges and the disease progresses, consistent with our OncoPrint results that ATM and TP53 alterations were most frequent in relapsed/refractory (R/R) MCL. We demonstrated that protein arginine methyltransferase-5 (PRMT5) was upregulated in R/R MCL, which predicted a poor prognosis. PRMT5 inhibitors displayed profound antitumor effects in the mouse models of MCL with mutated ATM and/or TP53, or refractory to CD19-targeted CAR T-cell therapy. Genetic knockout of PRMT5 robustly inhibited tumor growth in vivo. Co-targeting PRMT5, and ATR or CDK4 by using their inhibitors showed synergistic antitumor effects both in vitro and in vivo. Our results have provided a rational combination therapeutic strategy targeting multiple PRMT5-coordinated tumor-promoting processes for the treatment of R/R MCL with high mutation burdens.

The Role of New 3D Pathology and Lymphocyte Expression of Interstitial Inflammation in Pediatric-Onset Lupus Nephritis.

Lupus nephritis (LN) is a common and severe manifestation of pediatric-onset systemic lupus erythematosus (pSLE). It is one of the major causes of long-term glucocorticoid/immune suppressants use in pSLE. It causes long-term glucocorticoid/immune suppressants use and even end-stage renal disease (ESRD) in pSLE. It is now well known that high chronicity, especially the tubulointerstitial components in the renal biopsy, predicts a poor renal outcome. Interstitial inflammation (II), a component of activity in LN pathology, can be an early predictor for the renal outcome. With the advent of 3D pathology and CD19-targeted CAR-T cell therapy in the 2020s, the present study focuses on detailed pathology and B cell expression in II. We recruited 48 pSLE patients with class III/IV LN to analyze the risk of ESRD based on different II scores. We also studied 3D renal pathology and immunofluorescence (IF) staining of CD3, 19, 20, and 138 in patients with a high II score but low chronicity. Those pSLE LN patients with II scores of 2 or 3 showed a higher risk for ESRD (p = 0.003) than those with II scores of 0 or 1. Excluding patients with chronicity >3, high II scores still carried a higher risk for ESRD (p = 0.005). Checking the average scores from the renal specimens from different depths, the II, and chronicity showed good consistency between 3D and 2D pathology (interclass correlation coefficient [ICC], II = 0.91, p = 0.0015; chronicity = 0.86, p = 0.024). However, the sum of tubular atrophy plus interstitial fibrosis showed no good consistency (ICC = 0.79, p = 0.071). The selected LN patients with negative CD19/20 IF stains showed scattered CD3 infiltration and a different IF pattern of Syndecan-1 expression. Our study provides unique data in LN, including 3D pathology and different in situ Syndecan-1 patterns in LN patients.

15 - Fully Human CD19-Targeted CAR T-Cell Therapy for Relapsed or Refractory Large B-Cell Lymphoma: Results of a First-in-Human Phase I/II Study

15 - Fully Human CD19-Targeted CAR T-Cell Therapy for Relapsed or Refractory Large B-Cell Lymphoma: Results of a First-in-Human Phase I/II Study

Polatuzumab- Rituximab with or without Bendamustine As Second-Line Therapy for Relapsed Refractory Large B-Cell Lymphomas: Single Center Experience

Introduction: Polatuzumab-vedotin, a CD79 antibody drug conjugate, is approved in combination with bendamustine and rituximab (Pola-BR) in patients with relapsed or refractory (R/R) Large B-cell Lymphoma (LBCL) who have received at least 2 prior lines of therapy. However, the registrational phase Ib/II study by Dr. Sehn and colleagues (Sehn et al. J Clin Oncol 2019) included 11 patients treated with this regimen as second line (2L) therapy. Given these data, real world applications have included the use of 2L polatuzumab rituximab (Pola-R) combination, with or without bendamustine, either as stand-alone treatment or as a bridge to cellular therapy. Here, we sought to analyze institutional outcomes of LBCL patients treated with this regimen and to determine whether the pattern of failure to frontline chemoimmunotherapy (1L CIT) was associated with subsequent response to polatuzumab containing regimen. Methods: We retrospectively reviewed data on 22 patients who received Pola-R with or without bendamustine as 2L therapy for LBCL at Miami Cancer Institute as of January 31st, 2022. Response was assessed by investigators using the Lugano criteria (Cheson et al. J Clin Oncol 2014). Objective response rate (ORR) and complete response (CR) rates were compared between primary refractory (no CR to 1L CIT) and relapsed LBCL patients. Progression free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Results: Baseline characteristics are summarized in Table 1. The median age was 73 years (range, 57 to 92). Seven (32%) patients were ≥80 years old. The majority of patients (54%) had de novo DLBCL and non-GCB (59%) cell of origin by Hans algorithm. The median number of polatuzumab cycles was 4 (range, 1 to 7 cycles). Pola-BR was initiated with full dose bendamustine for 14 (64%) patients. Bendamustine was omitted for 6 (27%) patients and 2 (9%) patients received both Pola-R and Pola-BR cycles. The investigator-assessed best ORR was 68% (95% confidence interval [CI], 48.7% to 87.6%). Fourteen (64%) patients achieved a CR. Of the 6 patients who received Pola-R, 67% achieved a CR. The ORR was 90% for patients with relapsed disease, and 40% for those with primary refractory disease (p=0.057). CR rates were significantly lower in patients with disease refractory to 1L CIT (p=0.019). Median follow-up was 11 months. Nine (41%) patients received cellular therapy after polatuzumab containing regimen. Of these, 4 patients received CD19 targeted CAR T-cell therapy, and 5 patients received high dose chemotherapy followed by autologous stem cell transplant (ASCT). Other subsequent therapies after polatuzumab were: tafasitamab plus lenalidomide (n=1), loncastuximab tesirine (n=1) and conventional salvage CIT (n=3). Median PFS and OS were not reached. The 1-year PFS and OS were 54.5% (95% CI, 32.1% to 76.9%) and 70.5% (95% CI, 47.6% to 93.4%), respectively. Progression free survival and overall survival estimates are shown in Figure 1. Conclusions: Polatuzumab-Rituximab with or without bendamustine is an effective salvage option for R/R LBCL patients in the second-line setting. Refractoriness to frontline CIT was associated with lower response to subsequent polatuzumab treatment. Patients treated with this regimen can be successfully bridged to cellular approaches as definitive therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD22-Targeted CAR-Modified T-Cells Safely Induce Remissions in Children and Young Adults with Relapsed, CD19-Negative B-ALL after Treatment with CD19-Targeted CAR T-Cells

Introduction: Children and young adults with B-lymphoblastic leukemia (B-ALL) who relapse with CD19 antigen loss after CD19-targeted CAR T-cell therapy have a dismal prognosis, with a median overall survival of 9.7 months. Novel immunotherapies are urgently needed for this population. CD22 can serve as a potential alternative target. In our initial pilot trial of CD22-targeted/4-1BB CAR T cells, clinical activity was unexpectedly poor. Based on additional preclinical work demonstrating that shorter scFv linkers improve T-cell effector function, a novel CD22/4-1BB CAR construct with a short scFv linker (CART22-65s) was developed and investigated in a phase 1 study (NCT02650414). Methods: After fludarabine/cyclophosphamide lymphodepletion, patients (pts) were infused with CART22-65s using a 3-day fractionated dosing scheme with the 2nd/3rd doses held for early signs of cytokine release syndrome (CRS). The primary endpoint was safety. Key secondary endpoints were feasibility, efficacy, and CAR T cell expansion and persistence. Results: Nineteen pts were enrolled and had a product successfully manufactured. Of 17 pts infused (median age, 16 years; range 3-28 years), all had CD19-negative, relapsed disease after prior CD19-directed therapy (CART19, n=16; blinatumomab, n=1); 8 had prior inotuzumab; 9 had prior stem cell transplant (SCT); and 3 had multiple prior SCTs. Median bone marrow blasts immediately pre-infusion was 31% (range, 0-99%); 8 patients had >75% blasts. The median cell dose infused was 4x106 CART22-65s cells/kg (range, 0.8 to 10 x 106). In vivo cellular kinetics by qPCR demonstrated robust initial CAR T cell proliferation peaking at a median of 20 days post-infusion, and persistence that correlated with clinical response (Figure A). At day 28, 13/17 (77%) pts achieved a complete remission (CR) of which 10 (77%) had undetectable minimal residual disease (uMRD) by flow cytometry. One additional pt cleared MRD at month 2 without intervention. Notably, 4/5 (80%) pts refractory to prior inotuzumab achieved a CR with CART22-65s. Of 11 pts with a best response of uMRD CR, 5 proceeded to consolidative SCT, including 3 with prior SCT: 2 remain in CR post-SCT, 2 are in CR after a brief course of alternative therapy for emergence of MRD, and 1 had a CD22+ relapse post-SCT. Of 6 pts with uMRD CR who did not proceed to SCT (5 had a prior SCT), 5 experienced relapsed disease with a range of CD22 expression and 1 remains in CR 30 months after infusion with ongoing CAR T persistence. Of 2 pts with MRD+ CR (1 CD22+, 1 CD22-), both progressed to morphologic relapse; one received alternative therapy prior to progression. Of 4 pts with non-response, all died of disease within 9 months. Thus, with a median follow-up of 29 months, median relapse-free, event-free, and overall survival were 5.3 (95% CI, 1.9-NR), 5.8 (95% CI, 2.1-NR), and 16.5 (95% CI, 9.3-NR) months, respectively (Figure B). In addition, 2 patients received CART22-65s retreatment for CD22+ relapsed disease after initial infusion. Both engrafted, but only 1 achieved uMRD CR. CRS was observed in 15 pts (grade 1, n=10; grade 2, n=5), and mild neurotoxicity in 6 pts (grade 1, n=5; grade 2, n=1). In the retreatment setting, 1 pt developed grade 3 CRS, which resolved, and grade 3 encephalopathy/hypotonia, but died of progressive disease prior to resolution. Autopsy findings were consistent with leukomyelopathy of the spinal cord, which was inconclusive in its relation to CART22-65s. Other SAEs included: (1) platelet refractoriness and recurrent, severe inflammatory reactions to platelet transfusions in 1 pt that improved with corticosteroids and anakinra; and (2) delayed hemophagocytic lymphohistiocytosis in 1 pt after CART22-65s retreatment that resolved without intervention. Conclusion: CART22-65s induced remissions in 77% of children and young adults with highly refractory, CD19-negative B-ALL, including in 4/5 pts refractory to prior CD22-directed therapy. Manufacturing was successful, and the overall toxicity profile was favorable even in pts with high disease burden. Several uncommon toxicities were observed, but the only grade 3 CRS and neurotoxicity events occurred in the retreatment setting. Our observation of high initial response rates, but later recurrences, highlights the need to use CART22-65s as part of combination therapy. These promising preliminary data provide support for a combined CD19/22 approach using the CART22-65s construct. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Ongoing Pilot Study of Targeted Radioimmunotherapy (131-I Apamistamab) Conditioning Prior to CD19-Targeted CAR T-Cell Therapy for Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia or Diffuse Large B-Cell Lymphoma

An Ongoing Pilot Study of Targeted Radioimmunotherapy (131-I Apamistamab) Conditioning Prior to CD19-Targeted CAR T-Cell Therapy for Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia or Diffuse Large B-Cell Lymphoma

18FDG PET/CT Parameters for the Prediction of CAR T-Cell Therapy Response among Patients with Large B-Cell Lymphoma

<h3>Purpose/Objective(s)</h3> To define the prognostic value of quantitative positron emission tomography/computed tomography (PET/CT) parameters in diffuse large B-cell lymphoma (DLBCL) patients undergoing CAR T-cell therapy. <h3>Materials/Methods</h3> Sixty-three patients who had PET-CT scans done pre-CAR T infusion were retrospectively identified and analyzed in a single institution database of DLBCL patients treated with commercially available CD19-targeted CAR T-cell therapy (axi-cel, tisa-cel). The predictive and prognostic value of low vs. high metabolic tumor volume (MTV), total lesion glycolysis (TLG), and SUV were analyzed with cutoff values determined by the sample mean for MTV and TLG and mode for SUV. The response evaluation was conducted based on the post-CAR T best overall response, which was defined as the lowest disease burden measured on the PET scan at any time after CAR T-cell therapy. Univariate analysis was conducted using logistic regression, while the multivariate analysis was performed using logistic regression and Cox proportional hazards models, controlling for age ≥60, gender, CAR T product, and receiving bridging therapy. <h3>Results</h3> The median follow-up was 10.7 months (range, 0.6 - 46.5 months). The best objective overall response (complete response-CR and partial response) and CR rates post-CAR T were 78% (n=49) and 54% (n=34), respectively. On univariate analysis, low pre-CAR T TLG and MTV were predictive of CR post-CAR T (OR=4.7, p=0.01, OR=8.03, p=0.001, respectively). Patients with high pre-CAR T TLG or MTV had an increased risk of mortality (OR=3.6, p=0.03, OR=4.04, p=0.02), and a high pre-CAR T TLG was associated with a non-significant trend of increased grade 3-4 neurological events (OR=2.8, p=0.2). There was also a trend to shorter OS among patients with high SUVmax pre-CAR T (8.3 months vs. 24.4 months, p=0.3). On multivariate analysis, bulky disease (≥5 cm) (HR=6.08, 95% CI: 2.6-14.2, p=< 0.001), high MTV (HR=2.4, 95% CI: 1.13-4.9, p=0.022), and high TLG (HR=2.35, 95% CI: 1.1-5.0, p=0.026) were significantly associated with inferior OS. <h3>Conclusion</h3> High pre-CAR T MTV and TLG values are significantly associated with inferior CR and OS rates and a trend toward higher risk of severe neurotoxicity among patients with LBCL treated with CAR T-cell therapy. Bulky disease (≥ 5 cm) is associated with significantly inferior OS. These data suggest that there may be a role for pre- CAR T radiation therapy targeting bulky site(s) with high FDG avidity, regardless of symptoms, thereby reducing disease burden prior to CAR-T cell therapy, which may lead to improved efficacy and reduced CAR T-related adverse events.

Characterization of Leukemic Resistance to CD19-Targeted CAR T-cell Therapy through Deep Genomic Sequencing.

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has been a clinical breakthrough for pediatric B-cell acute lymphoblastic leukemia (B-ALL), and loss of the CD19 target antigen on leukemic cells represents a major mechanism of relapse. Previous studies have observed CD19 mutations specific to CD19- relapses, and we sought to clarify and strengthen this relationship using deep whole-exome sequencing in leukemic cells expanded in a patient-derived xenograft. By assessing pre-treatment and relapse cells from 13 patients treated with CAR T-cell therapy, 8 of whom developed CD19- relapse and 5 of whom developed CD19+ relapse, we demonstrate that relapse-specific single-nucleotide variants and small indels with high allele frequency combined with deletions in the CD19 gene in a manner specific to those patients with CD19- relapse. Before CAR T-cell infusion, one patient was found to harbor a pre-existing CD19 deletion in the context of genomic instability, which likely represented the first hit leading to the patient's subsequent CD19- relapse. Across patients, preexisting mutations and genomic instability were not significant predictors of subsequent CD19- relapse across patients, with sample size as a potential limiting factor. Together, our results clarify and strengthen the relationship between genomic events and CD19- relapse, demonstrating this intriguing mechanism of resistance to a targeted cancer immunotherapy.