CD19-targeted Chimeric Antigen Receptor T-cell Research Articles

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.

Safety and efficacy of autologous and allogeneic humanized CD19-targeted CAR-T cell therapy for patients with relapsed/refractory B-ALL

BackgroundMurine chimeric antigen receptor T (CAR-T) cell therapy has demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). However, the potential immunogenicity of the murine single-chain...

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

Peripheral blood cellular profile at pre-lymphodepletion is associated with CD19-targeted CAR-T cell-associated neurotoxicity.

Infusion of second generation autologous CD19-targeted chimeric antigen receptor (CAR) T cells in patients with R/R relapsed/refractory B-cell lymphoma (BCL) is affected by inflammatory complications, such as Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Current literature suggests that the immune profile prior to CAR-T infusion modifies the chance to develop ICANS. This is a monocenter prospective study on 53 patients receiving approved CAR T-cell products (29 axi-cel, 24 tisa-cel) for R/R-BCL. Clinical, biochemical, and hematological variables were analyzed at the time of pre-lymphodepletion (pre-LD). In a subset of 21 patients whose fresh peripheral blood sample was available, we performed cytofluorimetric analysis of leukocytes and extracellular vesicles (EVs). Moreover, we assessed a panel of soluble plasma biomarkers (IL-6/IL-10/GDF-15/IL-15/CXCL9/NfL) and microRNAs (miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p) which are associated with senescence and inflammation. Multivariate analysis at the pre-LD time-point in the entire cohort (n=53) showed that a lower percentage of CD3+CD8+ lymphocytes (38.6% vs 46.8%, OR=0.937 [95% CI: 0.882-0.996], p=0.035) and higher levels of serum C-reactive protein (CRP, 4.52 mg/dl vs 1.00 mg/dl, OR=7.133 [95% CI: 1.796-28], p=0.005) are associated with ICANS. In the pre-LD samples of 21 patients, a significant increase in the percentage of CD8+CD45RA+CD57+ senescent cells (median % value: 16.50% vs 9.10%, p=0.009) and monocytic-myeloid derived suppressor cells (M-MDSC, median % value: 4.4 vs 1.8, p=0.020) was found in ICANS patients. These latter also showed increased levels of EVs carrying CD14+ and CD45+ myeloid markers, of the myeloid chemokine CXCL-9, as well of the MDSC-secreted cytokine IL-10. Notably, the serum levels of circulating neurofilament light chain, a marker of neuroaxonal injury, were positively correlated with the levels of senescent CD8+ T cells, M-MDSC, IL-10 and CXCL-9. No variation in the levels of the selected miRNAs was observed between ICANS and no-ICANS patients. Our data support the notion that pre-CAR-T systemic inflammation is associated with ICANS. Higher proportion of senescence CD8+ T cells and M-MDSC correlate with early signs of neuroaxonal injury at pre-LD time-point, suggesting that ICANS may be the final event of a process that begins before CAR-T infusion, consequence to patient clinical history.

Cellular kinetics and outcome of tisagenlecleucel for diffuse large B-cell lymphoma

CD19-targeted chimeric antigen receptor T-cell (CAR-T) therapy has shown promise as treatment of relapsed or refractory B-cell malignancies. However, the clinical utility of early CAR-T monitoring within 1 month after infusion has not been elucidated. In this study, we quantitatively measured CAR-T kinetics in peripheral blood on days 2, 4, 7, 9, 11, 14, 21, and 28 post-infusion using flow cytometry and quantitative polymerase chain reaction in 13 patients with relapsed refractory diffuse large B-cell lymphoma (DLBCL) treated with tisagenlecleucel (tisa-cel). No relationships were identified between bulk CAR-T kinetics and treatment outcomes. Interestingly, the magnitude of CD4+ CAR-T expansion was higher in responders than in nonresponders, while CD8+ CAR-T expansion was minimal in responders. Additionally, CAR-T proliferation was more pronounced in patients with cytokine release syndrome. Our results suggest that CD4+ CAR-T cellular kinetics within 1 month after CAR-T infusion may predict its efficacy after tisa-cel therapy in adult patients with DLBCL.

Extracellular Vesicles Expressing CD19 Antigen Improve Expansion and Efficacy of CD19-Targeted CAR-T Cells

CAR-T cell therapy is effective in the treatment of certain hematological malignancies, and the expansion and functional persistence of CAR-T cells in vivo are crucial to clinical efficacy. The aim of this study was to investigate the potential of extracellular vesicles (EVs) modified with the CAR antigen to promote the efficacy of CAR-T cells in vivo. We generated HEK293T-derived EVs to present the CD19 antigen as the CAR target. In vitro, EVs expressing CD19 antigen (CD19 EVs) were co-incubated with anti-CD19 CAR-T cells. Then, proliferation, cytokine secretion, CD107a expression, tumor killing, subsets, and immune checkpoint expression were measured to assess CAR-T cell function. After infusion of CD19 EVs pretreated CAR-T cells into a lymphoma xenograft mouse model, flow cytometry and digital PCR were used to measure the expansion of CAR-T cells, and tumor volumes were continuously monitored to assess the anti-tumor efficacy of CAR-T cells in vivo. Another mouse model was created to investigate the effect of in vivo injection of CD19 EVs on the functional persistence of CAR-T cells, and safety was determined by histopathology of the main organs. CD19 EVs activated CAR-T cells in an antigen-specific and dose-dependent manner and promoted the selective expansion and cytokine secretion of co-cultured CAR-T cells. Specifically, CD19 EVs preferably increased the expansion of the CAR-T subpopulation with a high surface CD19-CAR density and consequently enhanced the anti-tumor activity of CAR-T cells. Futhermore, CD19-EVs-primed CAR-T cells achieved superior proliferation and anti-tumor effects in a mouse model with lymphoma xenograft. In vivo administration of CD19 EVs promoted the functional persistence of CAR-T cells in the xenograft mouse model. Our findings indicate that antigen-expressing EVs can be utilized as a boost to improve CAR-T cell efficacy in vitro and in vivo.

Whole body FDG PET/MR for progression free and overall survival prediction in patients with relapsed/refractory large B-cell lymphomas undergoing CAR T-cell therapy

BackgroundTo find semi-quantitative and quantitative Positron Emission Tomography/Magnetic Resonance (PET/MR) imaging metrics of both tumor and non-malignant lymphoid tissue (bone marrow and spleen) for Progression Free Survival (PFS) and Overall Survival (OS) prediction in patients with relapsed/refractory (r/r) large B-cell lymphoma (LBCL) undergoing Chimeric Antigen Receptor (CAR) T-cell therapy.MethodsA single-center prospective study of 16 r/r LBCL patients undergoing CD19-targeted CAR T-cell therapy. Whole body 18F-fluorodeoxyglucose (FDG) PET/MR imaging pre-therapy and 3 weeks post-therapy were followed by manual segmentation of tumors and lymphoid tissues. Semi-quantitative and quantitative metrics were extracted, and the metric-wise rate of change (Δ) between post-therapy and pre-therapy calculated. Tumor metrics included maximum Standardized Uptake Value (SUVmax), mean SUV (SUVmean), Metabolic Tumor Volume (MTV), Tumor Lesion Glycolysis (TLG), structural volume (V), total structural tumor burden (Vtotal) and mean Apparent Diffusion Coefficient (ADCmean). For lymphoid tissues, metrics extracted were SUVmean, mean Fat Fraction (FFmean) and ADCmean for bone marrow, and SUVmean, V and ADCmean for spleen. Univariate Cox regression analysis tested the relationship between extracted metrics and PFS and OS. Survival curves were produced using Kaplan–Meier analysis and compared using the log-rank test, with the median used for dichotomization. Uncorrected p-values < 0.05 were considered statistically significant. Correction for multiple comparisons was performed, with a False Discovery Rate (FDR) < 0.05 considered statistically significant.ResultsPre-therapy (p < 0.05, FDR < 0.05) and Δ (p < 0.05, FDR > 0.05) total tumor burden structural and metabolic metrics were associated with PFS and/or OS. According to Kaplan-Meier analysis, a longer PFS was reached for patients with pre-therapy MTV ≤ 39.5 ml, ΔMTV≤1.35 and ΔTLG≤1.35. ΔSUVmax was associated with PFS (p < 0.05, FDR > 0.05), while ΔADCmean was associated with both PFS and OS (p < 0.05, FDR > 0.05). ΔADCmean > 0.92 gave longer PFS and OS in the Kaplan-Meier analysis. Pre-therapy bone marrow SUVmean was associated with PFS (p < 0.05, FDR < 0.05) and OS (p < 0.05, FDR > 0.05). For bone marrow FDG uptake, patient stratification was possible pre-therapy (SUVmean ≤ 1.8).ConclusionsMTV, tumor ADCmean and FDG uptake in bone marrow unaffected by tumor infiltration are possible PET/MR parameters for prediction of PFS and OS in r/r LBCL treated with CAR T-cells.Trial registrationEudraCT 2016–004043-36.

Computational model of CAR T-cell immunotherapy dissects and predicts leukemia patient responses at remission, resistance, and relapse

BackgroundAdaptive CD19-targeted chimeric antigen receptor (CAR) T-cell transfer has become a promising treatment for leukemia. Although patient responses vary across different clinical trials, reliable methods to dissect and predict patient...

Timing of PD-L1 Blockade with Durvalumab May Affect Outcomes of CD19 CAR-T Cell Therapy for Relapsed/Refractory Large B-Cell Lymphoma

Introduction CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown high overall response rates (ORRs) in patients (pts) with relapsed/refractory (R/R) large B-cell lymphoma (LBCL); however, CD19 CAR-T cells fail to elicit a complete response (CR) in a considerable proportion of pts. PD-L1 expression in the tumor microenvironment could impair the efficacy of CAR-T cell therapy. PD-1/PD-L1 pathway blockade may enhance the function and antitumor activity of CAR-T cells. Here, we report the analysis of a phase I dose-finding study (NCT02706405) of combination therapy with CD19-targeted 4-1BB-costimulated CAR-T cells (JCAR014) and escalating doses of durvalumab (durva), an anti-PD-L1 monoclonal antibody, in adults with R/R LBCL. Methods Pts were treated in 1 of 2 groups. All pts received lymphodepletion (LD) with cyclophosphamide (Cy) and fludarabine (Flu) followed by JCAR014 infusion. Pts in group 1 (Grp 1) received the first dose of durva (225 mg or 750 mg) after JCAR014 infusion. Pts in group 2 (Grp 2) received the first dose of durva (7.5 mg, 22.5 mg, 75 mg, 225 mg, or 750 mg) 1 day prior to JCAR014 infusion. Up to 10 doses of durva were administered at 4-week intervals after JCAR014 at the highest identified safe dose until toxicity or disease progression. Adverse events (AEs) were graded by CTCAE 4.03, except for cytokine release syndrome (CRS), which was graded according to Lee 2014 consensus criteria. Response was reported according to PET/CT 2014 Lugano criteria. Results Between 01/23/2017 and 07/02/2020, 34 pts were screened, 32 underwent leukapheresis, and 29 were enrolled and received LD and JCAR014. Eleven pts were treated in Grp 1 and 18 in Grp 2 (Table 1). All pts received 2 x 106 JCAR014 cells/kg, except for the first 2 pts treated on the study who received 7 x 105 cells/kg. The most common AEs of any grade related to LD and JCAR014 with or without durva were CRS (41%), neutropenia (21%), neurotoxicity (17%), and hypogammaglobulinemia (17%). The incidences of any grade and grade ≥ 3 CRS and neurotoxicity were similar between Grp 1 and Grp 2 (Table 2A). Patients in Grp 2 had later onset of CRS after JCAR014 infusion compared with Grp 1 (median of 6 vs. 4 days, P = .05) and shorter duration of CRS (median of 3 vs. 8 days, P = .08). Among the 27 pts evaluable for dose-limiting toxicity (DLT), 2 (7%) had DLT: 1 pt with grade 4 CRS in Grp 2 (durva 225 mg) and 1 pt with prolonged grade 3 neurotoxicity in Grp 2 (durva 750 mg). No maximum-tolerated dose of durva was identified. In pts evaluable for response (n = 26), the ORR and the CR rate at 3 months were 35% (95% CI, 17-56) and 27% (95% CI, 14-46). We retrospectively compared the response rates of LBCL pts treated with JCAR014 in combination with durva (NCT02706405) with LBCL pts who received the same Cy/Flu lymphodepletion regimen and JCAR014 dose (2 x 106 cells/kg) without durva (JCAR014 alone cohort) on our previous phase I/II clinical trial (NCT01865617). There was a trend towards lower ORR (P = .08) and CR rate (P = .09) in pts treated with JCAR014 in combination with durva compared with those treated in the JCAR014 alone cohort (Table 2B). There were no significant differences in ORR and CR rate between patients in Grp 1 and those who received JCAR014 alone. However, despite a trend towards lower tumor burden, pts in Grp 2 (first durva before JCAR014) had lower ORR (P = .07) and CR rate (P = .03) compared with those treated in the JCAR014 alone cohort, and a trend towards lower CR rate compared with those treated in Grp 1 (first durva after JCAR014; P = .16). Patients treated with JCAR014 alone had significantly shorter time to CAR-T cell peak counts in blood compared with patients treated with JCAR014 in combination with durva (median of 8 vs. 14 days, P = .01). We did not observe significant differences in peak CAR-T cell expansion, area under the curve (AUC) from day 0 to 28 after JCAR014 infusion, and day 28 CAR-T cell counts by qPCR between the cohorts. Conclusion The combination of JCAR014 with durva for the treatment of adult pts with LBCL was safe. Our findings suggest that the timing of initiation of durva therapy is a key variable that may affect outcomes. To our knowledge, this is the first report suggesting that PD-L1 blockade may impact toxicity and antitumor response in pts with LBCL undergoing CD19 CAR-T cell therapy. Additional studies will be required to determine the optimal approach for combining CD19 CAR-T cell therapy with PD-1/PD-L1 pathway blockade in LBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Reduced Intensity Reinduction Followed By Blinatumomab Achieves Excellent MRD Clearance with Reduced Toxicity and Facilitates Timely Transplant in Children and Young People with Relapsed B-Precursor Acute Lymphoblastic Leukemia

Introduction Despite the emergence of effective novel immunotherapies, the outcome of relapsed pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains poor due to treatment-related mortality (TRM) and resistant disease. In response to accumulating evidence on the efficacy and low toxicity of blinatumomab in relapsed B-ALL and the availability of highly active CD19-targeted chimeric antigen receptor T-cell therapy (CART), we redesigned the United Kingdom (UK) relapse treatment pathway, based on two key guiding principles: 1) to reduce chemotherapy burden and associated toxicity through a move away from the highly intensive ALL R3 backbone towards increased reliance on blinatumomab to clear disease, and 2) to identify treatment failures early and direct them to CART, given the poor responses reported with hematopoietic stem cell transplant (HSCT) in this context. Methods Patients were treated with UKALL induction chemotherapy (<16yr: 3-drugs; >16yr: 4-drugs) until adequate blast clearance. Patients with blasts <5% at Day 15 progressed directly to blinatumomab; remaining patients continued to Day 28. Following reinduction, patients received up to 2 cycles of blinatumomab as a single agent. HR patients (early/very early relapses and/or HR cytogenetics) proceeded to HSCT once minimal residual disease (MRD) <0.01%. Those with high-risk (HR) isolated central nervous system (CNS) disease had the option to proceed directly to HSCT following CSF blast clearance without blinatumomab. All Standard risk (SR) patients continued on chemotherapy alone. Patients who were refractory after re-induction (>25% disease), blinatumomab (MRD >0.01%), or for whom there was no available donor were considered for CART. All patients were required to be discussed at a national tumour board, and data were subsequently collected every 3 months for analysis. Events were defined as death, relapse, and secondary cancer. Results Between September 2018 and July 2022, 111 patients were treated on the pathway; 63 (56.8%) were HR and 48 (43.2%) were SR. There were 26 patients >15yr, 3 Infant ALL, 2 Ph+ ALL and 4 with Down Syndrome. Induction therapy achieved disease reduction to <5% and CNS clearance in 98 patients (88%). Of the 90 patients with BM involvement, 52.2% required only 15 days of induction therapy. Induction failure (>25% blasts) occurred in 6.3%, comparable to the 7.1% using the R3 induction in the COG AALL1331 trial (p=1.00); 6/7 patients with induction failure successfully underwent CART infusion. The rate of end of induction MRD <0.01% in the HR group was also similar to COG AALL1331 (30% vs. 27.4%, p=0.669). Importantly, there were no cases of TRM with the reduced intensity induction, compared to 2.4-4% reported with the ALL R3 induction (p=0.06). Ninety patients received blinatumomab, of whom 87% achieved a complete MRD response, with grade 3 or 4 treatment-related toxicity in 11 patients (12.2%). Amongst HR patients, 86.8% underwent HSCT, significantly more than in the blinatumomab-treated IR/HR arm of COG AALL1331 (66%, p=0.02). With a median follow-up of 12 months (range 2-49), 19% of HR patients and 8.3% of SR patients have relapsed, comparable to results achieved in the blinatumomab-treated arm of the COG AALL1331 trial. There were no deaths in the SR group, and 12 deaths in the HR group (6 due to relapse/refractory disease, 6 due to post-HSCT TRM). Conclusions Our findings demonstrate that a reduced intensity reinduction can achieve equivalent disease control to the more intensive ALL R3 induction, with a substantial reduction in TRM. Although follow-up is short, consolidative blinatumomab achieved very high rates of MRD clearance, allowing a greater number of HR patients to reach potentially curative therapy with HSCT, the key predictor of outcome in the COG AALL1331 trial. We propose that our strategy represents a new standard of care for reinduction therapy in relapsed childhood B-ALL. DO and AV are joint senior authors Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Low Incidence of Fungal Infections after Chimeric Antigen Receptor T-Cell Therapy for Non-Hodgkin Lymphoma in an Endemic Region for Coccidioidomycosis

Introduction: CD19-targeted chimeric antigen receptor T-cell therapy (CAR-T) has revolutionized the treatment of non-Hodgkin lymphomas (NHL). Due to underlying NHL, CAR-T therapy, and treatment of associated toxicities, CAR-T recipients are immunocompromised and, consequently, at high risk of infection. Evidence informing prophylaxis (PPx) is limited and often varies based on institutional practice. This study sought to determine the incidence of fungal infection after CAR-T in patients with NHL treated in Arizona and describe PPx strategies. Methods: Using Mayo Clinic Arizona's (MCA), Banner MD Anderson's (BMDACC), and Banner University Medical Center's (BUMC) CAR-T databases, patients who received commercial CD19-targeted CAR-T for NHL between September 2018 and June 2022 were identified. Disease characteristics, prior and subsequent therapy, antimicrobial use, and infectious disease testing were collected. Early and late adverse events including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), hypogammaglobulinemia, and cytopenias were collected. The 2020 European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium consensus definitions were used to define probable and proven invasive fungal disease (IFD). Results: A total of 55 patients from MCA met inclusion criteria (Table 1). The median age at infusion was 60 years (range 19-78) and median follow-up time was 9.6 months (range 1.3-46.0) post CAR-T infusion. Most patients were male (78.2%, n=43), had diffuse large B-cell lymphoma (45.5%, n=25), and received axicabtagene ciloleucel (69.1%, n=38). Early and late toxicities (Table 2) included grade ≥3 CRS (7.2%, n=4), grade ≥3 ICANS (25.5%, n=14), immunoglobulin G <400 mg/dL (41.8%, n=23) and a CD4 T-cell count <200 cells/mm^3 (81.1%, n=30). Thirty-six patients (65.4%) received tocilizumab, 29 patients (52.7%) received steroids for CRS and/or ICANS. At MCA, two patients (3.6%) had mucocutaneous Candida infection, at day 10 and day 14 post-infusion, respectively. Four patients (7.3%) developed IFD, all of whom were in partial or complete response. Two (3.6%) had biopsy proven pulmonary IFD caused by Aspergillus and Coccidioides at day 64 and day 233 post-infusion, respectively. Two (3.6%) had probable pulmonary IFD caused by Aspergillus and Pneumocystis jirovecii at day 107 and day 209 post-infusion, respectively. These two patients died shortly after their IFD diagnosis, however also had proven COVID-19 infection and bacterial sepsis, respectively. Of those with IFD, only the patient with Pneumocystis jirovecii pneumonia (25%, n=1/4) had received steroids for the treatment of CAR-T related toxicity. No IFDs were identified by database review at Mayo Clinic in Rochester or Florida, thus leading our group to consider endemic risk factors in Arizona. At BUMC, 3 patients (8.5%, n=3/35) developed pulmonary IFD, 2 with Aspergillus and 1 with Coccidioides. At BMDACC, 1 patient (3%, n=1/37) developed Saccharomyces of the tongue at day 30 while receiving fluconazole. At MCA, antifungal PPx, most commonly with fluconazole, was used in those with neutropenia lasting >7 days (49%, n=27), whereas it was used routinely at BMDACC and BUMC for at least 180 days. All patients received trimethoprim-sulfamethoxazole PPx for at least 180 days unless an alternative was necessary. Patients at MCA, BUMC, and BMDACC are routinely screened for Coccidioides prior to CAR-T, although prior anti-B-cell therapy may impact these results. Serum Coccidioides antibody, complement fixation, and immunodiffusion were positive in only 1 MCA patient prior to receiving CAR-T and this patient had an uncomplicated course, although received extended PPx fluconazole. Conclusions: Fungal infection after CAR-T for NHL appears to be uncommon. IFD tends to occur later, often beyond the typical time frame of antifungal PPx and/or caused by organisms not necessarily prevented by typical PPx regimens. Future studies are needed to individualize infectious disease screenings, monitoring, and antifungal PPx in this population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase I Clinical Trial of Point-of-Care Manufactured Fresh Anti-CD19/20/22 Chimeric Antigen Receptor T Cells for Treatment of Relapsed or Refractory Lymphoid Malignancies (Non-Hodgkin Lymphoma, Acute Lymphoblastic Leukemia, Chronic Lymphocytic Leukemia, B Prolymphocytic Leukemia)

Background: CD19-targeted CAR-T cells have transformed the treatment of relapsed/refractory B-cell malignancies. However, relapse occurs in over 50% of patients treated with commercial CD19 directed CAR-T due to antigen escape from selective immune pressure either on the target antigen or due to exhaustion and lack of persistence of CAR-T cells. Hence there is an unmet need for next-generation CAR-Ts that have broad antigen recognition and novel costimulatory domains to promote survival and decrease exhaustion of CAR-Ts. Targeting three B-cell antigens simultaneously is a novel approach to prevent antigen-negative relapse while inclusion of the OX40 domain will promote CAR-Ts persistence and expansion. Whether targeting three commonly expressed antigens is feasible or safe and will result in durable remissions is not known. We hypothesize that this trispecific targeting of CD19/20/22 CAR-T construct will be safe and effective in patients with relapsed, refractory B-cell malignancies. Study Design and Methods: This parallel group phase 1 study follows a 3+3 phase 1 design to determine the safety of the infusion of point-of-care manufactured fresh CAR-T cells targeting CD19/20/22 and to find the recommended phase II dose for this cellular therapy. CAR-T cells will be manufactured over 6 days. Patients will be enrolled in one of the two cohorts: Cohort A (single infusion): Non-Hodgkin's Lymphoma (NHL) with lesions <= 5 cm; Indolent lymphomas; B-PLL (B-Pro lymphocytic leukemia); Chronic Lymphocytic Leukemia -CLL (without Richter's transformation), Cohort B (split infusion): B-ALL (B-Acute lymphoblastic leukemia); Lymphoid blast crisis of Chronic myeloid leukemia; CLL (with Richter's transformation); NHL with lesions > 5 cm and/or lymphoblastic lymphoma, or NHL with circulating lymphoma cells. Patients who are CAR-naïve will be enrolled in this study. The study involves infusion of anti-CD19/20/22 CAR-T cells administered after an immunosuppressive conditioning regimen of fludarabine and cyclophosphamide. Cyclophosphamide 60 mg/kg will be infused intravenously over 60 minutes on day -6. Fludarabine 25 mg/m2 will be infused intravenously daily over 30 minutes on days -5 to -3. In each group, patients will be enrolled sequentially to each dose level, starting with dose level 1. Three CAR-T dose-levels: 5x105, 1x106 & 2x106 cells/kg/dose will be explored to determine MTD. For cohort B patients, the total CAR-T cell dose infused, will be divided in two doses, dose 1 (day 0) is 40% of the total dose and dose 2 (day 7) is 60% of total dose. The minimum number of subjects enrolled in the complete study will be 18 (9 per cohort) and the maximum number in dose escalation will be 36 (18 per cohort). Patients in each cohort will be monitored for 30 days before enrollment of next patientTwo patients have been enrolled in the trial and received fresh infusion of Anti CD19/20/22 CAR-T cells and accrual is ongoing. Updates on the initial cohort of patients will be presented at the meeting. ClinicalTrials.gov NCT: NCT05418088 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD19-Targeted CAR-T Cells in Refractory Systemic Lupus Erythematosus: Safety, Efficacy, and Mechanistic Insights from the First Five Patients

Background: Systemic lupus erythematosus (SLE) is an auto-immune disease that is based on dysregulation of the adaptive immune system, autoantibody production and immune complex-mediated tissue damage. While treatment of SLE has substantially improved, a subset of patients experiences severe progressive disease despite T- and B-cell targeted therapy. Aim: For patients with refractory SLE, we aimed to achieve deep depletion of B cells using autologous CD19-CAR T cells with the goal of achieving an immunological reset and drug free remission. Methods: Five patients (4 women, 1 men) with SLE refractory to several immune-suppressive drugs including pulsed steroids, hydroxychloroquine, mycophenolate, cyclophosphamide, intravenous immunoglobulins, rituximab, and belimumab were treated with CD19 CAR T cells in a compassionate use program. Mean patient age was 21.8 ± 2.3 years, mean disease duration was 4.6 ± 3.0 years and mean SLE disease activity index (SLEDAI) was 11.8±3.8. Autologous T cells were transduced with a lentiviral anti-CD19 CAR vector composed of the FMC63 scFv, a CD8-derived hinge region, TNFRSF19-derived transmembrane domain, CD3ζ intracellular domain, and 4-1BB co-stimulatory domain and expanded within the local GMP facility utilizing the closed automated CliniMACS Prodigy® system (Miltenyi Biotec, Bergisch Gladbach, Germany). CAR T cells were re-infused at a dose of 1x106 CAR T cells/kg body weight after lymphodepletion with fludarabine/ cyclophosphamide. All SLE treatments with the exception of low dose prednisolone was stopped before CAR-T cell administration. After CAR-T cell treatment, no other immune-modulatory or immune-suppressive treatment was given. Tolerability was assessed following international guidelines. Results: Despite long-lasting high dose steroid treatment, sufficient numbers of CD19 CAR-T cells could be generated from 5/5 SLE patients. Lymphodepletion led to expected reduction of white blood cells. Following lymphodepletion, CAR T cells expanded in vivo with a peak on day 9 and a deep depletion of B cells was achieved in all patients. Bone marrow toxicity towards non-B cells was short and all patients recovered neutrophils (>500/nL) before day 14 post CAR T cells. Only mild CRS of °I occurred in all five patients. One patient received a single dose of tocilizumab due to persisting fever >24 hours. There were no ICANS, no CRS of any organ, and no infections. Remission of SLE according to DORIS criteria was achieved in all 5 patients after 3 months and the mean (±SD) SLEDAI score after 3 months was 0.4±0.9. Laboratory parameters normalized in all SLE patients after CAR-T transfer including seroconversion of anti-dsDNA antibodies and all patients were free of immune-suppressive drugs by month 3. One patient presented with proteinuria at month 4. Kidney biopsy as well as serum diagnostics showed no signs of SLE. After a steroid pulse, proteinuria went back to normal. Despite reconstitution of B cells no relapse of SLE was observed in the follow up of any of the patients which are now 17, 12, 8, 7, and 5 months after CAR T cell therapy. Reappearing B cells were naïve and showed non-class switched B cell receptors. While pre-existing vaccine-specific antibodies towards measles or VZV persisted, several auto-reactive antibodies were abrogated. Quality of life was poor at decision to treatment and improved substantially at month three. Improvement was mostly driven by abrogation of joint pain and of fatigue. Conclusions: Taken together, the preliminary data from this pilot study show that CD19 CAR T-cell therapy is very well tolerated in patients with a non-malignant B cell derived autoimmune disease and induce rapid remission of severe refractory SLE. Longer follow-up and higher patient numbers from controlled clinical trials are needed to confirm these promising results.

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

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

A Phase 2/3, Randomized, Double Blind, Placebo-Controlled, Multicenter Study of NKTR-255 Vs Placebo Following CD-19 Directed CAR-T Therapy in Patients with Relapsed/Refractory Large B-Cell Lymphoma

Background: Autologous T cells engineered to express a CD19-directed chimeric antigen receptor (CAR) have shown high overall response rates in treatment-refractory CD19+ B-cell non-Hodgkin lymphoma (NHL). However, over half of these patients will have lymphoma that fails to achieve remission or will relapse; thus, strategies to further improve the long-term efficacy of CAR-T cell products are needed. NKTR-255 is a novel investigational polymer-conjugated IL-15 agonist, designed to activate, proliferate and expand natural killer (NK) and CD8+ T-cells in vivo. NKTR-255 also promotes the survival and expansion of memory CD8+ T cells. Preclinical data in B-cell lymphoma xenograft models have shown that NKTR-255 enhanced expansion, survival, and anti-tumor activity of human CD19 CAR-T cells. Further, clinical studies have demonstrated that NKTR-255 promoted CD8+ T cell expansion in patients with relapsed/refractory (r/r) NHL who previously received CAR-T cell therapy (NCT04136756). Here, we describe a planned Phase 2/3 clinical trial of NKTR-255 following CAR-T cell therapy. Methods: This is a phase 2/3, randomized, double blind, placebo-controlled, multicenter study of NKTR-255 vs placebo following CD-19 directed CAR-T therapy. Eligible patients who have r/r large B-cell lymphoma and are planned for treatment with an FDA-approved CAR-T product (axi-cel or liso-cel) will be enrolled. All patients will receive initial study drug administration of NKTR-255 intravenously, starting approximately 14 days following CAR-T therapy, with continued dosing every 21 days. The study will be comprised of two stages: 1) Phase 2: patients will be randomized to placebo or one of multiple dose cohorts of NKTR-255 (Stage 1) and 2) Phase 3: patients will be randomized to placebo or the selected NKTR-255 dose regimen (Stage 2). The primary objective for Stage 1 is to identify the dose of NKTR-255 for the Phase 3 part of the study based on the safety and tolerability of NKTR-255 as well as the complete response rate (CRR) at month 6. The primary efficacy endpoints of Stage 2 are CRR at month 6 and event-free survival. Efficacy analyses will be performed separately for Stage 1 and Stage 2. Efficacy, as measured by PET-CT, will be evaluated according to the Lugano Criteria (Cheson 2014). The key eligibility criteria following CD19 targeted CAR-T cell infusion include no grade ≥ 1 cytokine release syndrome (CRS) on the day of NKTR-255 administration, no grade ≥ 3 CRS within 72 hours proceeding NKTR-255 administration, no grade ≥ 3 immune effector cell-associated neurotoxicity (ICANS) of > 72 hours duration, no grade ≥ 2 ICANS on the day of NKTR-255 infusion and no tocilizumab and/or dexamethasone within 48 hours proceeding NKTR-255 administration. Conclusions: Based on preclinical and clinical evidence, NKTR-255 has the potential to improve efficacy of currently approved cellular therapies. This trial evaluates safety and efficacy of NKTR-255 following commercial CD19 CAR-T therapy to enhance antitumor effect and durability of responses. 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

Cytokine Release Syndrome and Neurotoxicity after Chimeric Antigen Receptor T-Cell Therapy for Relapsed/Refractory B-Cell Lymphoma: Is There an Association with Bridging Radiotherapy?

<h3>Purpose/Objective(s)</h3> CD19-targeting chimeric antigen receptor T-cell (CART) therapy is an effective treatment for relapsed/refractory aggressive B-cell lymphoma (r/rABL). However, cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) may limit applicability and typically require hospitalization for toxicity management. Bridging radiotherapy (RT) may be used to debulk tumor burden or maintain performance status prior to CART therapy infusion, but associations with CRS and ICANS remain unclear. We assessed associations of bridging RT with CRS and ICANS among a cohort of patients (pts) receiving commercial CART therapy. <h3>Materials/Methods</h3> 94 pts receiving tisagenlecleucel (n=66) or axicabtagene ciloleucel (n=28) for r/rABL between April 2018 and June 2020 at a single institution were retrospectively identified. 22 pts (23%) received bridging RT (B-RT group) and 72 (77%) did not (NB-RT group). CRS was graded with ASTCT and ICANS with CTCAE v5.0. Grade ≥2 CRS (CRS2) and grade ≥2 ICANS (ICANS2) were compared between B-RT and NB-RT groups using Fisher exact test. Logistic regression was used to assess associations of patient characteristics, tumor bulk metrics (metabolic tumor volume [MTV], largest lesion max diameter [LLMD], and total lesion glycolysis [TLG]), and bridging therapy with CRS2 and ICANS2. <h3>Results</h3> B-RT group had better performance status (ECOG ≥1 41% vs 68%, p=0.027) and numerically but not significantly higher MTV (median 42.6mL vs 15.2, p=0.38), LLMD (median 5.8cm vs 3.5, p=0.12), and TLG (median 298.3 mL*SUV vs 60.8, p=0.22). Median B-RT dose was 31 Gy (IQR 20-40), most commonly in 2-2.5 Gy/fraction (n=17, 77%). Grade 1, 2, and 3 CRS occurred in 27 (29%), 19 (20%), and 5 (5%) pts, respectively. Grade ≥2 CRS occurred in 4/22 pts (18%) in B-RT group vs 20/72 pts (28%) in NB-RT group (p=0.16). Univariate analysis revealed associations between the following and CRS2: MTV (odds ratio [OR] 1.02 per 10 mL, p=0.025), LLMD (OR 1.12, p=0.01), largest lesion ≥5 cm (OR 2.98, p=0.046) and TLG (OR 1.02 per 100 mL*SUV, p=0.014), but not B-RT (OR 0.58, p=0.37), bridging systemic therapy (B-ST) (OR 1.33, p=0.56) or ECOG ≥1 (OR 1.33, p=0.56). Grade 1, 2, 3, and 4 ICANS occurred in 11 (12%), 6 (6%), 5 (5%), and 2 (2%) pts, respectively. Grade ≥2 ICANS occurred in 1/22 pts (5%) in B-RT group vs 12/72 pts (17%) in NB-RT group (p=0.29). Univariate analysis revealed associations between the following and ICANS2: MTV (OR 1.02 per 10 mL, p=0.048), TLG (OR 1.02 per 100 mL*SUV, p=0.017), and a trend for ECOG ≥1 (OR 3.98, p=0.085), but not B-RT (OR 0.24, p=0.18) or B-ST (OR 1.22, p=0.75). <h3>Conclusion</h3> In this single-institution study, bridging RT was used for good performance status pts with bulky tumors and was associated with low rates of CRS and ICANS. An ongoing multi-institutional effort will help refine predictive models for these toxicity endpoints and further elucidate the role of bridging RT.

Radiotherapy as a Bridging Approach in High-Grade B-Cell Lymphomas before CD19-Targeted CAR T-Cell Therapy

<h3>Purpose/Objective(s)</h3> To evaluate the safety and efficacy of bridging radiation therapy (RT) in patients with relapsed/refractory high-grade B cell lymphomas (HGBCL) undergoing CD19-targeted chimeric antigen receptor (CAR) T-cell therapy. <h3>Materials/Methods</h3> A multi-institutional retrospective study was performed for 352 patients with HGBCL treated with CAR T-cell therapy. 140 of 352 patients (40%) received bridging therapy prior to CAR T infusion: systemic therapy alone in 98 patients, and RT +/- systemic therapy in 42 patients. This report focuses on the outcome of the 42 patients who received bridging RT. Overall survival (OS) from the date of CAR T infusion was estimated with the Kaplan-Meier method. The time to in-field progression was defined as the time from the start date of bridging RT until the time of in-field progression. <h3>Results</h3> The median follow-up from the date of CAR T infusion was 5.2 months (range, 9 days-33.6 months). Thirty-four patients (81%) received RT within 30 days of CAR T infusion, while 8 patients (19%) received RT at least 30 days before infusion. The most common indication for bridging RT was symptomatic local progression, with 16 patients starting RT prior to apheresis due to clinical urgency. Sites of RT include: head and neck (12), central nervous system (8), chest (5), pelvis (5), abdomen (4), extremities (2), paraspinal area (3), and multiple sites (3). The median dose/fractionation were 24 Gy (range, 8-37.5 Gy) and 8 fractions (range, 2-15 fractions). Three patients (7%) had in-field progression at a median time of 3.2 months (range, 1.2-4.6 months), all of whom had bulky disease (≥5 cm) at the time of apheresis. Superior median OS was observed among the 38 patients (90%) who achieved objective in-field responses after bridging-RT (25.6 months vs. 2.1 months; p=<0.001). Only 2 patients experienced grade 3-4 RT-related toxicities. <h3>Conclusion</h3> Our data supports that RT is an effective bridging approach for patients with locally progressive HGBCL prior to CAR T-cell therapy. It is associated with limited toxicity, and offers excellent in-field control in the immediate post-CAR T timeframe. Patients with in-field response to bridging RT have significantly better survival outcome.

Radiation Therapy for Bridging and Improving CAR-T Cell Therapy

<h3>Purpose/Objective(s)</h3> CD19-targeted chimeric antigen receptor (CAR) T cells have transformed the treatment of patients with relapsed or refractory CD19-positive hematologic malignancies. However, a significant subset of these patients either fails to respond or eventually relapses. Moreover, patients who are candidates for CART-19 therapy often have symptomatic disease that requires some form of treatment to support them during the manufacture period. An ideal bridging therapy would simultaneously both (1) sensitize the tumor to CART-19 attack to increase antitumor control and (2) debulk the disease during manufacture. Radiotherapy (RT) is an established curative and palliative cancer treatment regimen, with approximately half of cancer patients with solid tumors receiving RT sometime during their disease. Several reports have underscored the existence of threshold doses (and regimens) that are able to switch on different damage programs that profoundly affect responses to therapy by eliciting potent immune modulatory effects, prompting immunologically mediated tumor cell death. These effects have been attributed to multiple pathways, including the activation of the cGMP-cAMP Synthetase (cGAS), Stimulator of Interferon Genes (STING) pathway with Type I Interferons expression, and tumor associated antigen (TAA) cross priming with anti-tumor CD8⁺ T cell elicitation, ultimately inducing "abscopal" effects. By virtue of the immunomodulatory effects of RT, we hypothesize that RT could serve as a successful bridging strategy for CAR T-cell therapy. <h3>Materials/Methods</h3> To investigate this hypothesis, we established the A20 Lymphoma CART-19 therapy mouse model in our lab and used it to perform preliminaries experiments. A20 cells were implanted into both flanks of the animals. Mimicking human immune adjuvant doses used in our institute for RT bridging, 20 days after tumor cell implantation, we radiated one of the two tumors using a dose of 8Gy divided in two fractions of 4Gy each. Twenty-four hours later 1 × 10ep6 CART-19 cells were infused intravenously. <h3>Results</h3> While the effects of RT in combination with CART-19 have similar impact on the tumor growth of the irradiated mass, a significant increase of the antitumor effects of the single therapy was observed on the non-irradiated tumor. FACS studies demonstrated heavy leukocyte infiltration with increased presence of infused CART-19 as well indigenous T cells. To investigate possible molecular mechanisms involved in the increased effects on the non-irradiated tumor, we performed a qPCR array and observed increased expression of genes linked to cross priming, including IFN type I and Batf3. A20 cells express the ectopic retroviral antigen gp70 with well characterized AH1 immune-dominant epitope. AH1 tetramer staining on infiltrating T cells demonstrated increased staining in tumor from mice treated with the combination. <h3>Conclusion</h3> Overall these results suggest RT may serve as optimal debulking and bridging therapy for CAR-T cell therapy.