Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this resistance remain elusive. Using a genome-wide loss-offunction screen, we identifi ed that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnifi ed by the induction of CAR T-cell functional impairment. These fi ndings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fi tness. Our fi ndings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction. SIGNIFICANCE:Resistance to CART19 is a signifi cant barrier to effi cacy in the treatment of B-cell malignancies. This work demonstrates that impaired death receptor signaling in tumor cells causes failed CART19 cytotoxicity and drives CART19 dysfunction, identifying a novel mechanism of antigenindependent resistance to CAR therapy.

Unintentional transduction of B-cell acute lymphoblastic leukemia blasts during CART19 manufacturing can lead to CAR19+ leukemic cells (CARB19) that are resistant to CART19 killing. We developed an anti-CAR19 idiotype chimeric antigen receptor (αCAR19) to specifically recognize CAR19+ cells. αCAR19 CAR T cells efficiently lysed CARB19 cells in vitro and in a primary leukemia-derived xenograft model. We further showed that αCAR19-CART cells could be used as an “antidote” to deplete CART19 cells to reduce long-term side effects, such as B-cell aplasia.

Chimeric Antigen Receptor T-cell (CART) immunotherapy led to unprecedented responses in patients with refractory/relapsed B-cell non-Hodgkin lymphoma (NHL); nevertheless, two-thirds of patients fail this treatment. Resistance to apoptosis is a key feature of cancer cells that associates with treatment failure. In 87 NHL patients treated with anti-CD19 CART, we found that chromosomal alteration of BCL-2, a critical anti-apoptotic regulator, in lymphoma cells was associated with reduced survival. Therefore, we combined CART19 with the FDA-approved BCL-2-inhibitor, venetoclax, and demonstrated in vivo synergy in venetoclax-sensitive NHL. However, higher venetoclax doses for venetoclax-resistant lymphomas resulted in CART toxicity. To overcome this limitation, we developed venetoclax-resistant CART by overexpressing mutated BCL-2(F104L) which is not recognized by venetoclax. Notably, BCL-2(F104L)-CART19 synergized with venetoclax in multiple lymphoma xenograft models. Furthermore, we uncovered that BCL-2 overexpression in T cells per se enhanced CART anti-tumor activity in preclinical models and in patients by prolonging CART persistence.

BackgroundThe characteristics of patients undergoing atrial fibrillation (AF) ablation and subsequent outcomes in community practice are not well described.Methods and ResultsUsing the Outcomes Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF), we investigated the prevalence and impact of catheter ablation of AF. Among 9935 patients enrolled, 5.3% had previous AF ablation. Patients with AF ablation were significantly younger, more frequently male, and had less anemia, chronic obstructive pulmonary disease, and previous myocardial infarction (P<0.05 for all analyses) than those without previous catheter ablation of AF. Ablated patients were more likely to have a family history of AF, obstructive sleep apnea, paroxysmal AF, and moderate-to-severe symptoms (P<0.0001 for all analyses). Patients with previous ablation were more often in sinus rhythm on entry into the registry (52% vs. 32%; P<0.0001). Despite previous ablation, 46% in the ablation group were still on antiarrhythmic therapy. Oral anticoagulation was prescribed in 75% of those with previous ablation versus 76% in those without previous ablation (P=0.5). The adjusted risk of death (hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.52 to 1.18; P=0.2) and cardiovascular (CV) hospitalization (HR, 1.06; 95% CI, 0.90 to 1.26; P=0.5) were similar in both groups. Patients with incident AF ablation had higher risk of subsequent CV hospitalization than matched patients without incident ablation (HR, 1.67; 95% CI, 1.24 to 2.26; P=0.0008).ConclusionsIn U.S. clinical practice, a minority of patients with AF are managed with catheter ablation. Subsequent to ablation, there were no significant differences in oral anticoagulation use or outcomes, including stroke/non–central nervous system embolism/transient ischemic attack or death.Clinical Trial RegistrationURL: http://www.clinicaltrials.gov. Unique identifier: NCT01165710.

Chimeric Antigen Receptor T cells (CART) have led to unprecedented clinical responses in relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphomas (NHL), and multiple myeloma. However, despite these exciting results, most patients treated with CART therapy either do not respond or eventually relapse. Moreover, CART therapy has not yet been proven effective in several hematological malignancies, such as T cell lymphoma and leukemia (T-NHL/T-ALL) and acute myeloid leukemia (AML). Thus, there is a need to enhance currently available CART products and also to develop next-generation CART therapies to successfully treat additional neoplasms like T-NHL /T-ALL and AML. To this goal, we studied the cysteine-rich scavenger receptor CD5, an attractive target for CART immunotherapy because of its dual role in malignant cells and normal T cells. In malignant cells, CD5 is expressed by ~90% of TCL cells, by ~15-20% of AML cells, and also by most cases of chronic lymphocytic leukemia and mantle cell lymphoma (MCL). Of note, promising results using a CD28-based anti-CD5 CART against T-NHL and T-ALL were reported at this meeting in 2019 (LaQuisa C. Hill #199). In T cells, CD5 is highly expressed and inhibits T cell receptor (TCR)-mediated activation through several mediators including SHP-1, CBL, CBL-B, and GRB2. Therefore, we hypothesized that the genetic deletion of CD5 in engineered T cells could potentially enhance their effector functions. First, we designed and screened six 4-1BB-costimulated anti-CD5 lentiviral CAR constructs designed to have high, medium, and low affinity for CD5. We then selected the lead CAR5 construct (high affinity, heavy to light light chain orientation) based on its superior anti-tumor function in vivo in NOD-SCID IL2Rgnull (NSG) mice engrafted with T-cell leukemia (Jurkat). Then, to further improve CART5 activity, we optimized a CD5 short-guide RNA and deleted CD5 in CART5 cells using CRISPR-Cas9. CD5 gene deletion was reproducibly efficient (95-100% by flow cytometry and TIDE) during manufacturing (6 donors). Interestingly, the growth rate of wild type (WT) CART5 was comparable to CD5 KO CART5 and the expression of CD5 in WT CART5 was reduced. However, at the end of manufacturing, CD5 KO CART5 had increased central memory T cells (33.0% vs. 18.4%) and reduced expression of activation/exhaustion markers (PD-1 4.4% vs. 14.8%, LAG3 13.1% vs. 55.9%) compared to WT CART5, potentially indicating that CD5 KO reduces CART5-CART5 fratricide during manufacturing. We then compared wild type (WT) CART5 to CD5 KO CART5 in vitro using several T-NHL/T-ALL, MCL, and AML models, including primary samples (Sezary cells, primary MCL cells, and CD5+ AML cells). Both WT and CD5 KO CART5 were highly effective in killing CD5+ malignant cells, but CD5 KO CART5 showed enhanced proliferation upon activation. In two xenograft models of T-cell leukemia (primary T-ALL and Jurkat), CD5 KO CART5 showed dramatically increased tumor control compared to WT (Fig.1A, median overall survival for WT= 62 days vs. CD5 KO=not reached, p = 0.006, Mantel-Cox). This enhanced anti-tumor effect was associated with increased expansion of CD5 KO CART5 in the peripheral blood (PB) compared to WT CART5. To test the hypothesis that deletion of CD5 could increase the anti-tumor effect of CART targeting antigens other than CD5, we knocked out CD5 in anti-CD19 CART cells and tested their function in a CD19+ B-ALL xenograft model (NALM6). Remarkably, CD5 KO CART19 displayed significantly enhanced anti-leukemia activity and PB expansion compared to WT (Fig.1B,C, p&lt;0.05, p = 0.001, Mantel-Cox). Finally, we aimed to define the mechanisms by which CD5 KO enhances CART anti-tumor efficacy. We analyzed the phosphorylation of multiple targets in T cells after 15 minutes of CAR stimulation. Remarkably, CD5 KO CART5 cells had higher (&gt;2fold) phosphorylation of several signaling proteins, including key regulators of T cell activation, migration, and survival compared to WT CART5 (Fig. 1D). To confirm that the CD5 pathway was indeed the mediator of this effect, we knocked out SHP-1 in CART19 cells using CRISPR/Cas9 and observed increased leukemia killing. In conclusion, we demonstrate that CRISPR-Cas9 KO of CD5 enhances the anti-tumor activity of CAR T cells by enhancement of CAR-mediated activation and proliferation. These findings support the development of CD5 KO CART products in early-phase clinical trials. Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria. Barta:Atara: Honoraria; Monsanto: Consultancy; Seattle Genetics: Honoraria, Research Funding; Janssen: Honoraria; Pfizer: Honoraria. June:Tmunity Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Ziopharm Oncology: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Bluesphere Bio: Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Membership on an entity's Board of Directors or advisory committees; Cellares: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; DeCART Therapeutics: Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Kiadis Pharma: Current equity holder in private company. Ruella:Abclon, BMS, NanoString: Consultancy; UPenn/Novartis: Patents & Royalties; Abclon: Consultancy, Research Funding.