CD19 Chimeric Antigen Receptor T-cell Therapy Research Articles

Combination of Ibrutinib with CD19 Chimeric Antigen Receptor (CAR) T Cells Suggests Greater Contribution to CAR T-Cell Efficacy Than Other Approved BTK Inhibitors in the Preclinical Setting

Background: CD19-directed CAR T-cell therapy has demonstrated remarkable clinical benefits for patients with B-cell malignancies, but impaired CAR T-cell fitness can result in lack of response or relapse. Rational combinations that enhance T-cell fitness may overcome primary and secondary resistance. Bruton tyrosine kinase inhibitors (BTKi), such as Ibrutinib (Ibr) have been shown to enhance CAR T-cell efficacy (Fraietta et al, Blood 2016;Liu et al, Cancer Sci 2020), but the precise mechanism remains unclear. In addition to BTK, Ibr potently inhibits IL-2-inducible tyrosine kinase (ITK), an important mediator of T-cell signaling. Ibr has been shown to enhance Th1 polarization through suppression of the Th2 differentiation pathway, thereby potentiating a cytotoxic effector response (Qin et al, J Immunother 2020). Consistent with this finding, the Th1 phenotype was more frequent in peripheral blood mononuclear cells of Ibr-exposed patients than in acalabrutinib (Acala)-exposed patients (Scarfò et al, Blood 2021). Therefore, we aimed to assess the potential impact of ITK inhibition (ITKi) on CD19 CAR T-cell function by comparing Ibr to other approved BTKi that display low or no ITKi activity (Estupiñán et al, Front Cell Dev Biol 2021). Methods: CD19-41bb CAR T cells (CAR19) were generated by lentiviral transduction of healthy donor T cells. BTKi additivity to CAR19 function was evaluated in a Luciferase (Luc)+ Ramos, BTKi resistant systemic tumor model in NSG mice co-treated with covalent BTKis Ibr, Acala, zanubrutinib (Zanu), and CAR T cells. Next, to evaluate the impact of ITKi on CAR19 fitness and cytotoxicity, repeated stimulation in an in vitro cytotoxicity assay was performed at low CAR19 effector-to-tumor ratio (1:20) to mimic higher tumor burden stress conditions in the presence of each BTKi, including non-covalent BTKi pirtobrutinib (Pirto) (500 nM). Tumor cells were replenished every 3 days and cytotoxicity was measured on day 7 by luciferase assay. For phenotypic characterization, CAR T cells were repeatedly stimulated by plate-bound anti-Whitlow linker every 3 days. Viability and proliferation were measured by cell counter. Flow cytometry (FACS) was performed on surface and intracellular T-cell phenotypic, activation, and exhaustion markers. Metabolic fitness (glycolysis and ATP production) was characterized using Seahorse (Van der Windt, 2012). Impact of Ibr on signal strength was evaluated in CD19-expressing Jurkat reporter cells (CAR-J19) with knock-in of mKate in frame with Nur77. Ibr was added to coculture of CAR-J19 and tumor cells and upregulation of Nur77 was measured by FACS. Downstream ITK signaling pathway was measured by Western blot. Results: Preliminary in vivo experiments demonstrated that co-administration of each BTKi with CD19 CAR T cells improved tumor control at day 26 (N=5 per group; P≤0.0004 vs CAR T cells alone). In serial killing experiments, addition of Ibr to the coculture increased proliferation (n=2; P<0.05), viability (n=2; P=0.01), and the cytotoxicity of CAR T cells against Ramos cells ( P=0.02 vs CAR19 alone) to a greater extent than other BTKis tested (Fig. 1A). Prolonged stimulation of CAR19 with Ibr led to downregulation of activation and inhibitory markers on CAR19 and maintenance of a naive T-cell phenotype (Fig. 1B) that was confirmed by increased metabolic fitness (n=5; P<0.05). Addition of Ibr to CAR-J19 resulted in a 72% reduction in CAR activation and reduced phosphorylation of ITK, PLCG, and Zap70. Additional data including the impact of ITK knockout on CAR-T cells will be presented. Conclusions: Our study expands on the hypothesized mechanism of action underlying the clinical benefit observed with Ibr in CD19 CAR T-cell-treated patients with chronic lymphocytic leukemia or mantle cell lymphoma (Liu et al, Front Immunol 2022; Wang et al, NEJM 2020). Our data suggest that Ibr enhances CAR T-cell fitness to a greater extent than both covalent and noncovalent BTKi that display reduced or no ITKi activity. Through ITKi, Ibr tempers CAR T-cell activation induced cell death, maintains a naive state and prevents premature exhaustion, thereby enabling more efficacious tumor cell killing over time. Together, these data support the further exploration of Ibr to enhance CD19 CAR T-cell efficacy.

2708. Infectious Complications Among CD19 Chimeric Antigen Receptor T-cell Recipients

Abstract Background Current data on infectious complications of chimeric antigen receptor (CAR) T-cell therapy are limited to single center retrospective cohort studies. Presented here is a subset of patients from a multicenter retrospective cohort study including recipients of CAR T-cell therapy at Duke University, University of North Carolina at Chapel Hill, and Stanford University. Methods In a retrospective cohort of 66 patients who received CD19 CAR T-cell therapy Duke University between January 1, 2018 and August 31, 2021 rates and characteristics of bacterial, viral, and fungal infections within the first year after CAR T-cell infusion are described. Demographic, baseline clinical, and outcome variables are compared between patients who developed infection and those who did not. Results Forty-nine total infections occurred in 29 (43.9%) patients within 1 year of CAR T-cell infusion. Patients who developed infection were more likely to have an underlying malignancy other than diffuse large B-cell lymphoma (34% vs 8%, p = 0.031), and to have developed immune effector cell-associated neurotoxicity syndrome (62% vs. 32%, p = 0.016). Viral (23/49, 46.9%) and bacterial (20/49, 40.8%) pathogens predominated and infections most often involved the bloodstream (19/49, 38.8%) and lung (18/49, 36.7%).There were no statistically significant differences between rates of bacterial and viral infection based on time since CAR T-cell infusion (≤30 days vs 30 – 90 days vs >90 days), though fungal infections only occurred after 90 days in patients with relapse of their underlying disease. Conclusion Infectious complications after CAR T-cell therapy are common within the first year of infusion. Bacterial and viral infections predominate while fungal infections are rare and occurred only in patients with relapsed disease. Disclosures Melody Smith, MD, MS, BMS: Advisor/Consultant Krista Rowe-Nichols, RN, MSN, AOCNS, Bristol Myers Squibb: Advisor/Consultant|Kite Pharmaceuticals: Advisor/Consultant|Kite Pharmaceuticals: Speaker Bureau Christopher Kelsey, MD, Colgate-Palmolive: Expert Testimony|Johnson and Johnson: Expert Testimony Taewoong Choi, MD, Janssen biotech: Honoraria Matthew McKinney, MD, ADC Therapeutics: Advisor/Consultant|ADC Therapeutics: Honoraria|Beigene: Grant/Research Support|Beigene: Honoraria|Epizyme: Advisor/Consultant|Genentech: Advisor/Consultant|Genentech: Grant/Research Support|Genentech: Honoraria|Gilead/Kite: Advisor/Consultant|Gilead/Kite: Honoraria|Incyte: Grant/Research Support|Novartis: Advisor/Consultant|Seagen, Inc.: Advisor/Consultant|Takeda: Advisor/Consultant|TG therapeutics: Advisor/Consultant

Methylated Whole Genome Cell-Free DNA Sequencing before Chimeric Antigen Receptor T-Cell Therapy for Large B-Cell Lymphoma Predicts Treatment Outcomes

Introduction More than half of patients with relapsed or refractory large B-cell lymphoma (R/R LBCL) will experience progressive disease after CD19 chimeric antigen receptor T-cell therapy (CART). Circulating tumor DNA profiling has been used to prognosticate patients before CART, but mainly has focused on identification of somatic genomic aberrations. Epigenetic changes, specifically aberrant DNA methylation, can alter transcriptional regulation of genes important for treatment resistance. Altered methylation features can be detected from cell-free DNA (cfDNA) in patients with LBCL and are associated with outcomes after frontline immunochemotherapy. In the current study, we used whole genome bisulfite sequencing (WGBS) to analyze the methylation landscape in cfDNA from patient plasma before CART and identified novel methylation signatures associated with poor treatment outcomes. Methods Patients with R/R LBCL who received standard-of-care CART with plasma samples drawn before lymphodepletion (LD) or at cell infusion were identified. The primary endpoint was 3-month overall response after CART. cfDNA was extracted from 3-5 ml of plasma and cfDNA libraries were prepared using a Zymo-Seq cell free WGBS kit. Each library was sequenced to 300 - 600 million read pairs at a read length of 150 bp, enabling an average CpG coverage of 7-13X per detected CpG. Unique alignment rate among all libraries were about or above 82%. Reads were adapter trimmed with Trim Galore and aligned to reference genome hg19 using Bismark. Methylation calling was performed by MethylDackel. Differentially methylated cytosines (DMC, FDR ≤0.05) and regions (DMR) from WGBS data with an absolute methylation difference of ≥ 0.1 between 3-month responders and non-responders were identified using DSS. Functional enrichment analysis of genes overlapped by DMRs was performed by gprofiler2. Results A total of 22 patients with plasma collected before LD (n=21) or at cell infusion (n=1) who received CART were identified. Median follow-up was 21.5 months (95% CI 12.5-NR). Three-month response was evaluable in 21 patients (1 pending evaluation). Overall response rate (ORR) was 77% (8 CR, 9 PR) and 55% (9 CR, 2 PR) at one- and 3-months post-CART, respectively. Median PFS was 6.5 months (95% CI 3.0-NR), and median OS was 57.7 months (95% CI 7.7-NR). Patient and disease characteristics are described in Table 1. Differential methylation analysis identified 22,815 DMCs, of which 1,043 were hypomethylated and 21,772 were hypermethylated in non-responders. The global average methylation values across 25 million cytosines were 0.807 and 0.786 for non-responders and responders, respectively (p=0.265). A total of 582 DMRs were identified, of which 34 were hypomethylated and 548 were hypermethylated in non-responders (Figure 1). Of these, 281 (48%) overlapped intergenic regions, 265 (46%) gene bodies, and 33 (6%) promoter regions. DMRs overlapping DNA repair gene PARP1 (areaStat 88.2), B-cell receptor signaling genes SYK (27.2), FYN (21.4), and PTPN6 (20.9), polycomb repressor complex genes JARID2 (25.2) and BCOR (21.4), tumor suppressor WT1 (47.1), immune mediators NRP1 (80.0), TRAF3 (56.7), PAK3 (28.4), IRAK2 (28.7), and TNFS14 (23.6), and non-coding RNAs MIR4520 (95.8) and VTRNA-2 (23.1) were hypermethylated in non-responders and IL17RA (-33.2) hypomethylated. Functional enrichment analysis on overlapped genes also identified common molecular functions involving guanyl nucleotide exchange factors (p=0.0007), GTPase regulation (p=0.001), and cGMP binding (p=0.007). Discussion This proof-of-concept study is the first to perform WGBS on pretreatment plasma from patients with R/R LBCL to associate methylation signatures with post-CART outcomes. We identified that non-responders were enriched in hypermethylated DMRs compared to responders, overlapping with genes important to lymphomagenesis, immune function, and GTP energy metabolism. Further work will expand this cohort, compare with matched-tissue to find tumor-specific markers, and identify candidate DMR targets for targeted bisulfite sequencing from pre- and post-treatment plasma samples to assess response. By identifying high-risk methylation signatures in the plasma of CART recipients, we may be able to identify patients to be prioritized for novel treatment strategies such as combination with hypomethylating agents to improve outcomes.

Total Body Irradiation Versus Chemotherapy-Only Conditioning in Autologous Hematopoietic Stem Cell Transplantation for Large B-Cell Lymphoma

Introduction Recent data suggest that compared with high-dose chemotherapy conditioning and autologous stem cell transplant (ASCT), CD19-chimeric antigen receptor (CAR) T-cell therapy as second-line therapy improves outcomes in patients with primary refractory or early relapsed large B-cell lymphoma (LBCL). However, more than half of patients will not attain a long-term remission with CAR T-cell therapy, and may subsequently be considered for ASCT, indicating an unmet need for alternative therapies including strategies to improve ASCT outcomes. Given that LBCL is radioresponsive, we evaluated whether total body irradiation (TBI)-based conditioning may improve outcomes compared to chemotherapy-only conditioning among LBCL patients who underwent peripheral blood ASCT at our institution. Methods We reviewed the records of adults with relapsed/refractory LBCL who underwent ASCT at Fred Hutch Cancer Center between 2012-2021. LBCL was categorized as de novo DLBCL, transformed DLBCL, and double-hit lymphoma (DHL). Patients with primary CNS lymphoma were excluded. Given that TBI is generally reserved for younger patients (≤ 60 years), we excluded patients in the chemo-only group older than the oldest patient in the TBI group (58.7 years) to reduce the bias due to age differences. The hazards for failure for progression-free survival (PFS) and for overall mortality (OM) were compared between TBI-based and chemo-only groups using Cox regression with adjustment for clinically important variables including age, sex, histology, LDH at ASCT, conditioning type, PET positivity at the time of ASCT and year of ASCT. LDH at ASCT, age and year of ASCT were modeled as continuous linear variables. Results Among 225 patients, we excluded 121 patients in the chemo-only group who were older than 58.7 years, leaving 104 patients for the analysis ( Table 1). A total of 48 (46%) patients received TBI-based and 56 (54%) received chemo-only conditioning. All patients in the TBI group received 12 Gy in 6-8 fractions. Most patients (n=46, 82.14%) in the chemo-only group received BEAM conditioning. Patients receiving TBI-based conditioning had more adverse features including male sex (72.9% vs 58.9%), DHL histology (31.3% vs 7.1%), and PET-positive disease at the time of ASCT (39.6% vs 23.2%). With a median follow-up of 51.2 months (range 1.3-134.2) among survivors, point estimates of PFS at 2 and 5 years were 74.3% and 66.3%, respectively among TBI patients and 68.5% and 64.0%, respectively among the chemo-only patients ( Figure 1). Time to engraftment was similar between the groups. On multivariable analysis, compared to chemo-only conditioning, TBI was not associated with improved PFS (TBI vs chemo-only: HR 1.02, 95%CI 0.45-2.31, P=0.96) or OS (HR 0.85, 95%CI 0.37-1.98, P=0.7). Predictors of worse PFS were PET positivity at the time of ASCT (HR 3.39, 95%CI 1.61-7.17, P < 0.01) and higher LDH (HR 1.0011, 95%CI 1.0002-1.0020, P=0.02). Similarly, both PET positivity (HR 3.12, 95%CI 1.47-6.63, P<0.01) and higher LDH (HR 1.0013, 95%CI 1.0003-1.0022, P <0.01) were associated with worse OS. Age, sex, histology and year of ASCT were not significant. Conclusions Despite worse prognostic factors, TBI-based conditioning yielded similar outcomes compared to chemo-only conditioning for patients with relapsed/refractory LBCL undergoing ASCT. These results confirm prior findings from a registry-based study. Although a proportion of patients may achieve a long-term remission with ASCT, other strategies are needed to improve ASCT outcomes.

Development and Validation of a Clinical Prediction Model for the Efficacy of CD19 CAR T-Cell Therapy in DLBCL

Currently, Autologous CD19 chimeric antigen receptor T-cell therapy (CART) has become the most important treatment modality for relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL) beyond conventional multi-line treatments. However, it is worth noting that over 60% of CD19 CAR-T recipients will eventually experience relapse. A substantial amount of clinical research has been conducted to explore the reasons for the ineffectiveness or relapse of CAR-T therapy. Based on a comprehensive analysis of clinical data, we selected various combinations of predictive factors and used the TRIPOD guidelines to construct a logistic regression model for predicting the efficacy of CD19 CAR-T therapy. This study was conducted as a retrospective cohort study, including patients with confirmed diagnosis of DLBCL who underwent CD19 CAR-T therapy and met the following criteria: (1) aged 18 years or older but not exceeding 80 years; (2) voluntarily participated in the study and signed an informed consent form. The exclusion criteria for this study were: (1) addition of other chemotherapy drugs or targeted drugs within 90 days before assessing the best response after infusion, except for those who achieved complete response (CR) or progressive disease (PD); (2) death caused by non-tumor progression factors within 90 days after infusion. After the inclusion and exclusion criteria, a total of 54 patients with DLBCL undergoing CD19 CART at Shanghai Tongji Hospital of Tongji University ultimately participated in this study from January 1, 2019, to April 31, 2023. The derivation cohort consisted of 45 patients enrolled in clinical trials (NCT02537977, NCT03154775, CTR20201986, CTR20200561). The median age of the patients in the derivation cohort was 60 years (IQR: 50-67 years). Out of the 45 participants, 25 (55.6%) were male, and 29 (64.4%) had the non-germinal center B-cell (non-GCB) subtype, 1 had grade 3-4 Cytokine Release Syndrome (CRS). The validation cohort consisted of 9 patients involved in real-world studies. The median age of the patients in the validation cohort was 55 years (IQR: 52.5-69 years). Out of the 9 participants, 5 (55.6%) were male, and 6 (66.7%) had the non-GCB subtype, 2 had grade 3-4 CRS. The demographic and clinical characteristics of the derivation cohort were similar to those of the validation cohort. Based on the best CD19 CART response at 90 days, we divided the derivation cohort into two groups, CR and non-CR. Then, we selected a series of candidate predictive factors from baseline data such as clinical features, genetic features, and laboratory features before CART (Table 1). The results of univariate and multivariate logistic regression analysis were used to select specific factors (Gender, ECOG, TP53) as model prediction indicators based on p-values (p<0.05). There was no significant correlation between these factors (p>0.1). A score nomogram (Model1) was plotted using the regression coefficients of each predictive factor as weights (Fig.1 A), C-index (Model1) =0.872 (Fig.1 C). To facilitate better clinical use, we also constructed a risk nomogram (Model2) to assess the degree of risk (Fig.1 D). Model2 considers three factors: Male, ECOG≥2, TP53 altered. If a person has not any of these risk factors, they are categorized as low risk. If there is one risk factor present, they are deemed to be at moderate risk, whereas having two or more risk factors classifies them as high risk. C-index (Model2) =0.845 (Fig.1 F). Finally, the model's clinical efficacy was validated using real-world CART-treated patients, and the C-index was calculated as Model1=0.875 (Fig.1 G), Model2=0.7 (Fig.1 H), confirming the clinical impact of the model. In conclusion, this study has successfully developed an easily implementable clinical prediction model. For the first time, we have used CART clinical studies as the derivation cohort and real-world results as the validation cohort. Our scoring system provides clinicians with accurate and objective efficacy information, helping them identify the appropriate population for CAR-T therapy in DLBCL.

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

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

Long-term follow-up of humanized and murine CD19 CAR-T-cell therapy for B-cell acute lymphoblastic leukemia

Objective: Murine CD19 chimeric antigen receptor T-cell (CAR-T) products have been approved for the treatment of refractory/relapsed (R/R) B-cell acute lymphocytic leukemia (B-ALL) ; moreover, humanized products are also undergoing clinical trials. This study aimed to explore the differences in safety and short- and long-term follow-up efficacy between humanized and murine CD19 CAR-T-cells for treating relapsed and refractory B-ALL. Methods: Clinical data of 80 patients with R/R B-ALL treated with CD19-targeted CAR-T-cells at the Union Hospital of Tongji Medical College of Huazhong University of Science and Technology between May 2016 and March 2023 were analyzed, which included 31 patients with murine CAR-T and 49 with humanized products. Results: The proportion of patients with cytokine-release syndrome (CRS) in the murine and humanized groups was 63.1% and 65.3%, respectively. Moreover, a higher proportion of patients suffered from severe CRS in the murine group than in the humanized CAR-T group (19.4% vs 8.2%, P=0.174). Furthermore, one patient per group died of grade 5 CRS. The incidence of grade 1-2 immune effector cell-associated neurotoxicity syndrome (ICANS) was 12.9% and 6.1%, respectively; severe ICANS were not observed. Among patients receiving murine CAR-T-cells, an overall response (OR) was observed in 74.2%. Conversely, the OR rate of patients receiving humanized CAR-T-cells was 87.8%. During the median follow-up time of 10.5 months, the median recurrence-free survival (RFS) of patients with murine CAR-T-cells was 12 months, which was as long as that of patients with humanized CAR-T-cells. The median overall survival (OS) were not reached in both groups. Of the 45 patients with a bone marrow burden over 20% at baseline, humanized CAR-T therapy was associated with a significantly improved RFS (43.25% vs 33.33%, P=0.027). Bridging transplantation was an independent factor in prolonging OS (χ(2)=8.017, P=0.005) and PFS (χ(2)=6.584, P=0.010). Common risk factors, such as age, high proportion of bone marrow blasts, and BCR-ABL fusion gene expression, had no significant effect on patients' long-term follow-up outcomes. Three patients reached complete remission after reinfusion of humanized CAR-T-cells. However, one patient relapsed one month after his second infusion of murine CAR-T-cells. Conclusions: The results indicate that humanized CAR-T therapy showed durable efficacy in patients with a higher tumor burden in the bone marrow without any influence on safety. Moreover, it could overcome immunogenicity-induced CAR-T resistance, providing treatment options for patients who were not treated successfully with CAR-T therapies.

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

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

Low toxicity and excellent outcomes in patients with DLBCL without residual lymphoma at the time of CD19 CAR T-cell therapy.

CD19 chimeric antigen receptor (CAR) T-cell therapy represents a breakthrough for patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), inducing sustained remissions in these patients. However, CAR T cells can result in significant toxicities. Preinfusion disease burden is associated with toxicities and outcomes after CAR T-cell therapy. We identified 33 patients with R/R DLBCL treated at 8 academic centers who had no detectable disease at the time of CAR T-cell therapy. The median time from leukapheresis to CAR T-cell infusion was 48 (19-193) days. Nine patients received axicabtagene ciloleucel, and 24 received tisagenlecleucel. There was no severe (grade ≥3) cytokine release syndrome, and only 1 patient developed severe neurotoxicity (grade 4). After a median follow-up of 16 months, 13 patients relapsed (39.4%) and 6 died (18.1%). One-year event-free survival and overall survival were 59.6% and 81.3%, respectively. Our findings suggest that, in patients with R/R DLBCL who have an indication for CAR T-cell therapy, treating patients in complete remission at the time of infusion is feasible, safe, and associated with favorable disease control. Further exploration in a larger clinical trial setting is warranted.

Outcomes among adult recipients of CD19 CAR T-cell therapy for Burkitt lymphoma.

7571 Background: Burkitt lymphoma (BL) is a rare, but highly aggressive B-cell lymphoma associated with especially poor outcomes in patients with relapsed/refractory disease. Multiple CD19 chimeric antigen receptor (CAR) T-cell products have been FDA approved to treat relapsed/refractory B-cell non-Hodgkin lymphoma in the second-line setting, however little is known about CAR T-cell responses in patients with BL. This multicenter study aims to assess real-world outcomes among patients with BL treated with CAR T-cell therapy across the United States. Methods: This multicenter, retrospective chart review was conducted by abstracting data from the medical charts of patients with BL who received CAR T-cell therapy. Demographic characteristics were assessed along with pre-CAR treatments, bridging therapies, infusion-related complications, CAR-T responses, and the use of post-CAR systemic therapy and/or hematopoietic stem cell transplant. Results: A total of 13 patients with relapsed/refractory BL received CAR T-cell therapy after a median of 3 prior therapies (range 1-6). Most patients received axi-cel (n = 8), although tisa-cel (n = 3) and liso-cel (n = 2) were also represented. The median age of these patients was 38 (range 24-68). A total of 6 (46.2%) had evidence of central nervous system involvement at diagnosis and 5 (38.5%) had a prior history of autologous stem cell transplant. The overall response rate 1 month after infusion was 69.2% with a complete response rate of 53.8%. All patients experienced grade 1-2 cytokine release syndrome (CRS). No one experienced grade ≥3 CRS, but 2 patients developed grade ≥3 immune effector cell-associated neurotoxicity syndrome (ICANS). Twenty-eight-day mortality was 23%, including one patient who died from grade 4 ICANS. Regarding long term data, only 4 (30.8%) patients sustained a complete response for more than 6 months. Median progression-free survival was 4.2 months (95% CI 28.2-257.6), and the 1-year overall survival rate was 61.5% (95% CI 0.56-0.67). Two (15.4%) patients received consolidative allogeneic stem cell transplants after CAR T-cell therapy, but both ultimately developed progressive disease. Conclusions: Relapsed/refractory BL is a rare and aggressive disease that remains challenging to treat despite the advent of CAR T-cell therapy. Further investigation is needed to determine the most effective management of these patients. We expect to include additional patients in this analysis over the coming months.

Developing CAR‐T‐sparing Radiotherapy ‐ early dosimetric results

Background/objective: Radiotherapy (RT) is an effective treatment for residual lymphoma following CD19 chimeric antigen receptor T-cell (CAR-T) therapy. However, there are concerns regarding the potential effect of RT on CAR-T, particularly early after infusion. We initiated a prospective protocol to explore the effect of different RT planning parameters and techniques on dose to the blood, and thus CAR-T cells, in order to develop a “CAR-T sparing RT” for early RT after CAR-T therapy. Methods: We analysed 33 RT treatment plans for 11 lesions in 7 patients. Each lesion was planned 3 times; (1) a conventional plan using volumetric modulated arc therapy (VMAT) technique, (2) a plan with optimisation for blood vessels in the region as well as blood-rich organs and bone marrow, and (3) same as plan 2 but delivered with Flattening Filter-Free (FFF) beams to increase dose rate. Plans were compared with regards to planning target volume (PTV) coverage, dose homogeneity, organs at risk (OAR) doses including blood vessels, dose rate and beam-on time. Results: Using CAR-T sparing techniques, the mean dose to major blood vessels was reduced by a mean of 12% compared to the conventional VMAT plan. The beam-on time was reduced by a mean of 60% due to the increase in dose rate. The blood-rich organ doses were not significantly affected. There was no detrimental effect on the overall quality of plans in terms of PTV coverage and dose homogeneity. The effect of different planning measures employed varied in different cases depending on the PTV volume, the anatomical location, and the proximity of blood vessels. Examples of clinical cases will be presented in the meeting. Conclusion: Reduction of radiation dose to the blood is possible using a variety of technical adjustments as well as reduction of the beam-on time. Further work continues to understand the relative contribution of the individual parameters and optimise RT in the post-CAR-T setting. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Cellular therapies, Radiation Therapy Conflicts of interests pertinent to the abstract. N. G. Mikhaeel Honoraria: Gilead, Educational meeting honoraria, Oct 2022

Real-world evidence of the safety and survival with CD19 CAR-T cell therapy for relapsed/refractory solid organ transplant-related PTLD.

The use of CD19 chimeric antigen receptor T-cell (CAR-T) therapy for relapsed/refractory solid organ transplantation (SOT)-related post-transplant lymphoproliferative disorder (PTLD) is not well studied. We conducted a multicentre, retrospective analysis of adults with relapsed/refractory SOT-associated PTLD. Among 22 relapsed/refractory SOT-PTLD patients, the pathology was monomorphic B cell. Prior SOTs included 14 kidney (64%), three liver (14%), two heart (9%), one intestinal (5%), one lung (5%), and one pancreas after kidney transplant (5%). The median time from SOT to PTLD diagnosis was 107 months. Pre-CAR-T bridging therapy was used in 55% of patients, and immunosuppression was stopped completely before CAR-T infusion in 64%. Eighteen (82%) patients experienced cytokine release syndrome: one (5%) each grade (G) 3 and G4. The immune effector cell-associated neurotoxicity syndrome was observed in 16 (73%) patients: six (27%) G3 and two (9%) G4. The overall response rate was 64% (55% complete response). Three patients (14%) experienced allograft rejection after CAR-T. The two-year progression-free survival and overall survival rates were 35% and 58%, respectively. Additionally, the achievement of CR post-CAR-T was strongly associated with survival. Collectively, the safety and efficacy of CD19 CAR-T therapy in relapsed/refractory SOT-related PTLD appeared similar to pivotal CAR-T data, including approximately one-third of patients achieving sustained remission.

Which one is better for refractory/relapsed acute B-cell lymphoblastic leukemia: Single-target (CD19) or dual-target (tandem or sequential CD19/CD22) CAR T-cell therapy?

CD19 chimeric antigen receptor (CAR) T-cell therapy has shown great success against B-cell acute lymphoblastic leukemia (B-ALL). Tandem and sequential CD19/CD22 dual-target CAR T-cell therapies have been developed to reduce the possibility of CD19-negative relapse; however, the superior strategy is still uncertain. This study screened 219 patients with relapsed/refractory B-ALL who were enrolled in clinical trials of either CD19 (NCT03919240) or CD19/CD22 CAR T-cell therapy (NCT03614858). The complete remission (CR) rates in the single CD19, tandem CD19/CD22, and sequential CD19/CD22 groups were 83.0% (122/147), 98.0% (50/51), and 95.2% (20/21), respectively (single CD19 vs. tandem CD19/CD22, P = 0.006). Patients with high-risk factors achieved a higher rate of CR in the tandem CD19/CD22 group than in the single CD19 group (100.0% vs. 82.4%, P = 0.017). Tandem CD19/CD22 CAR T-cell therapy was one of the significant favorable factors in the multivariate analysis of the CR rate. The incidence of adverse events was similar among the three groups. Multivariable analysis in CR patients showed that a low frequency of relapse, a low tumor burden, minimal residual disease-negative CR and bridging to transplantation were independently associated with better leukemia-free survival. Our findings suggested that tandem CD19/CD22 CAR T-cell therapy obtains a better response than CD19 CAR T-cell therapy and a similar response to sequential CD19/CD22 CAR T-cell therapy.

Functional Validation of the RQR8 Suicide /Marker Gene in CD19 CAR-T Cells and CLL1CAR-T Cells

Chimeric antigen receptor T cell therapy (CAR-T) is a novel treatment that has produced unprecedented clinical effects in patients with hematological malignancies. Acute adverse events often occur following adoptive immunotherapy. Therefore, a suicide gene is helpful, which is a genetically encoded mechanism that allows selective destruction of adoptively transferred T cells in the face of unacceptable toxicity. RQR8 is a gene that integrates CD34 and CD20 epitopes. In our study, we incorporated the suicide gene RQR8 into CAR-T cells, so it enabled rituximab to eliminate vector/transgene-expressing T cells via antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity. In this work, we explored the functionality of RQR8 CAR-T cells in vitro and in vivo. We believe that RQR8 as a safety switch will make CAR-T cell therapy safer and less costly.

New Onset of Tuberculosis Complicating FDG PET/CT Evaluation in Patient With Recent Chimeric Antigen Receptor T-Cell Therapy.

A 16-year-old adolescent girl with CD19 chimeric antigen receptor (CAR) T-cell therapy for acute lymphoblastic leukemia experienced new onset of the fever. 18F-FDG PET/CT studies acquired at 1 and 2 months, respectively, after CAR-T, showed foci of abnormal activity in the mediastinal lymph nodes not seen on the study before therapy. However, these foci of abnormal activity were later proven due to newly developed tuberculosis after CAR T-cell therapy.

A digital nanoplasmonic microarray immunosensor for multiplexed cytokine monitoring during CAR T-cell therapy from a leukemia tumor microenvironment model

The release of cytokines by chimeric antigen receptor (CAR) T-cells and tumor resident immune cells defines a significant part of CAR T-cell functional activity and patient immune responses during CAR T-cell therapy. However, few studies have so far precisely characterized the cytokine secretion dynamics in the tumor niche during CAR T-cell therapy, which requires multiplexed, and timely biosensing platforms and integration with biomimetic tumor microenvironment. Herein, we implemented a digital nanoplasmonic microarray immunosensor with a microfluidic biomimetic Leukemia-on-a-Chip model to monitor cytokine secretion dynamics during CD19 CAR T-cell therapy against precursor B-cell acute lymphocytic leukemia (B-ALL). The integrated nanoplasmonic biosensors achieved precise multiplexed cytokine measurements with low operating sample volume, short assay time, heightened sensitivity, and negligible sensor crosstalk. Using the digital nanoplasmonic biosensing approach, we measured the concentrations of six cytokines (TNF-α, IFN-γ, MCP-1, GM-CSF, IL-1β, and IL-6) during first 5 days of CAR T-cell treatment in the microfluidic Leukemia-on-a-Chip model. Our results revealed a heterogeneous secretion profile of various cytokines during CAR T-cell therapy and confirmed a correlation between the cytokine secretion profile and the CAR T-cell cytotoxic activity. The capability to monitor immune cell cytokine secretion dynamics in a biomimetic tumor microenvironment could further help in study of cytokine release syndrome during CAR T-cell therapy and in development of more efficient and safer immunotherapies.

Safety and efficacy of co-administration of CD19 and CD22 CAR-T cells in children with B-ALL relapse after CD19 CAR-T therapy

BackgroundCD19-targeted chimeric antigen receptor T-cell (CAR-T) therapy has shown remarkable efficacy in treating relapsed or refractory pediatric B-lineage acute lymphoblastic leukemia (B-ALL). However, poor results are obtained when the same product is reused in patients who relapse after CAR-T. Therefore, there is a need to explore the safety and efficacy of co-administration of CD19- and CD22-targeted CAR-T as a salvage second CAR-T therapy (CART2) in B-ALL patients who relapse after their first CD19 CAR-T treatment (CART1).MethodsIn this study, we recruited five patients who relapsed after CD19-targeted CAR-T. CD19- and CD22-CAR lentivirus-transfected T cells were cultured separately and mixed before infusion in an approximate ratio of 1:1. The total dose range of CD19 and CD22 CAR-T was 4.3 × 106–1.5 × 107/kg. Throughout the trial, we evaluated the patients’ clinical responses, side effects, and the expansion and persistence of CAR-T cells.ResultsAfter CART2, all five patients had minimal residual disease (MRD)-negative complete remission (CR). The 6- and 12-month overall survival (OS) rates were 100%. The median follow-up time was 26.3 months. Three of the five patients bridged to consolidated allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CART2 and remained in MRD-negative CR at the cut-off time. In patient No. 3 (pt03), CAR-T cells were still detected in the peripheral blood (PB) at 347 days post-CART2. Cytokine release syndrome (CRS) only occurred with a grade of ≤ 2, and no patients experienced symptoms of neurologic toxicity during CART2.ConclusionsMixed infusion of CD19- and CD22-targeted CAR-T cells is a safe and effective regimen for children with B-ALL who relapse after prior CD19-targeted CAR-T therapy. Salvage CART2 provides an opportunity for bridging to transplantation and long-term survival.Trial registration: Chinese Clinical Trial Registry, ChiCTR2000032211. Retrospectively registered: April 23, 2020.

Salvage radiotherapy for relapsed/refractory non‐Hodgkin lymphoma following CD19 chimeric antigen receptor T-cell (CART) therapy

CD19-targeting chimeric antigen receptor T-cell (CART) therapy is a promising treatment for relapsed/refractory non-Hodgkin lymphoma, but most patients experience post-CART progression. We describe our institutional experience of salvage radiotherapy (SRT) in this setting. Of 94 patients who received CART therapy from 2018 to 2020, 21 received SRT for post-CART progression. Patients were divided into two groups: locoregional disease (n=9 [43%], all disease encompassable within an RT field) and advanced disease (n=12 [57%]). Patterns of failure, progression-free survival (PFS), overall survival (OS), and toxicity were assessed. Median time from CART infusion to SRT was 4.0months (range, 0.6-11.5months). In the locoregional disease group, 8/9 patients (89%) were treated with comprehensive SRT to a median dose of 37.5Gy in a median of 15 fractions. In the advanced disease group, all patients (n=12) were treated with focal SRT to a median dose of 20.8Gy in a median of 5 fractions. Median follow-up post-SRT was 15.2months. In-field response was observed in 8/9 (89%) in the locoregional disease and 8/9 (89%) evaluable patients in the advanced disease groups. 17/18 evaluable patients (94%) patients experienced post-SRT progression, all with a distant component. Median OS was 7.4months; 21months for locoregional disease versus 2.4months for advanced disease (p=0.0002). Median PFS was 1.1month, and similarly poor regardless of group. No grade≥3 toxicities occurred. SRT post-CART therapy appears safe with encouraging in-field response but high rates of out-of-field progression, even for those presenting with locoregional disease, highlighting the need for integration of novel systemic agents.

Allogeneic CD34+ selected hematopoietic stem cell boost following CAR T-cell therapy in a patient with prolonged cytopenia and active infection.

Hematological toxicity (hematotoxicity) leading to peripheral cytopenias is a common long-term adverse effect following the use of CD19-chimeric antigen receptor (CD19-CAR) T-cell therapies. However, management remains unclear for patients whose cytopenias persist beyond 1month after CAR T-cell infusion. We present the case of a 21-year old who received CD19-CAR T-cell therapy for relapse following a haploidentical transplant. He developed hematotoxicity and consequently multiple life-threatening infections. We administered a CD34+ hematopoietic stem cell boost (HSCB) from his transplant donor, which led to hematopoietic recovery and resolution of his infections without any effect on the activity of CD19-CAR T cells. CD34+ HSCB can be a safe and effective option to treat hematotoxicity following CD19-CAR T-cell therapy.

Safety and Efficacy of Humanized Versus Murinized CD19 and CD22 CAR T-Cell Cocktail Therapy for Refractory/Relapsed B-Cell Lymphoma.

CD19 chimeric antigen receptor T-cell (CAR-T) therapy is efficacious for refractory/relapsed (R/R) B-cell hematological malignancies, yet relapse due to CD19 antigen escape remains a challenge. Our trial explored simultaneous targeting of multiple B-cell antigens as a therapeutic approach that may reduce the risk of relapse. We tested the safety and efficacy of CAR19/22 T-cell cocktail therapy including murinized and humanized products among patients with R/R aggressive B-cell lymphoma. In the group that received the humanized product, 11/12 (91.7%) patients achieved an objective response, including 9/12 (75%) complete responses (CRs) by day 28. The overall response rate and CR rate in the murinized group was 92.9% (13/14) and 42.9% (6/14), respectively. Nine of 12 (75%) patients in the humanized group maintained CR at month 3 following infusion, compared to 5/14 patients (35.7%) in the murinized group. Progression-free survival (PFS) was more favorable in the humanized compared to the murinized group. Most patients had mild cytokine release syndrome (CRS) (grade 1-2) in both groups. This study demonstrates that CAR19/22 T-cell cocktail therapy is safe and effective for R/R B-cell lymphoma and that patients treated with a humanized CAR-T exhibited better efficacy compared to patients treated with a murinized CAR-T therapy.