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Importance The choice of induction agent during rapid sequence intubation (RSI) of critically ill adults may affect clinical outcomes. Although ketamine and etomidate are frequently used for RSI, their comparative effectiveness remains controversial. Objective To compare the safety of etomidate vs ketamine for emergency RSI in critically ill adults. Design, Setting, and Participants This cohort study used a target trial emulation with observational data collected between March 1, 2022, and April 30, 2024, from 18 emergency departments across Brazil, with data from the Brazilian Airway Registry Cooperation. Adults who underwent RSI and received either etomidate or ketamine as a sole hypnotic agent were included; those with preintubation cardiac arrest or immediate postintubation transfer were excluded. Exposure Administration of etomidate or ketamine as the induction agent for RSI. Main Outcomes and Measures The primary outcome was 28-day in-hospital mortality. Secondary outcomes included 7-day in-hospital mortality, first-attempt intubation success, and major adverse events (new hemodynamic instability, severe hypoxemia, and cardiac arrest) within 30 minutes after intubation. Inverse probability of treatment weighting was used to adjust for confounding. Risk ratios (RRs) and risk differences (RDs) with 95% CIs were used to compare outcomes between groups. Results Among 1810 patients (median age, 64 years [IQR, 50-74 years]; 1048 men [57.9%]), 514 received ketamine and 1296 received etomidate. The median shock index was higher in the ketamine group than the etomidate group (0.81 [IQR, 0.65-1.01] vs 0.76 [IQR, 0.59-0.99]), and preintubation vasopressor use was more common in the ketamine group than the etomidate group (191 of 514 [37.2%] vs 391 of 1296 [30.2%]). Weighted 28-day mortality was higher with etomidate than ketamine (60.5% [95% CI, 57.2%-63.8%] vs 54.4% [95% CI, 45.0%-63.9%]; RR, 1.14 [95% CI, 1.03-1.27]; RD, 7.6% [95% CI, 2.0%-13.3%]). Seven-day mortality was also higher with etomidate than ketamine (35.2% [95% CI, 32.0%-38.3%] vs 30.1% [95% CI, 23.5%-36.7%]; RR, 1.19 [95% CI, 1.04-1.35]). New hemodynamic instability within 30 minutes after intubation was more frequent with ketamine (24.2% [95% CI, 20.4%-28.0%] vs 18.9% [95% CI, 16.7%-21.0%]; RR, 0.78 [0.64-0.95]). There were no statistically significant differences in the other secondary outcomes. Conclusions and Relevance In this cohort study of critically ill adults undergoing RSI, etomidate use was associated with higher in-hospital mortality at 7 and 28 days compared with ketamine. These findings highlight the need for definitive randomized clinical trials to compare both agents.

Background ‘Exception from informed consent’ research requires community consultation to ensure that the views of the local population have been considered. Using market research companies to distribute surveys across local populations is attractive because it ensures adequate response rates. It is not known whether individuals who respond to surveys are typical of the targeted communities. The purpose of this study is to compare the demographics of the survey respondents with that of the general population in the same area. Methods This is an analysis of data collected as part of the ‘Trauma Resuscitation with Group O Whole Blood or Products’ (TROOP) trial. The surveys used a commercial market research service to reach the communities of interest and consisted of 18 questions designed to evaluate respondents’ willingness to be enrolled in the TROOP trial and the demographics of survey respondents. Results Catchment populations varied from 0.5 to 9.4 million residents, with a total of 33.5 million. The racial and ethnic composition of the population (and the survey respondents, in brackets) was 44.8% (57.0%) white, 25.7% (13.4%) Hispanic, 12.7% (19.4%) black, 8.0% (2.5%) multiracial, 7.9% (4.4%) Asian, 0.7% (0.9%) Native American, and 0.2% (0.5%) Islanders. Of the male population (and survey respondents, in brackets), 12.2% (15.3%) were aged 18years to 24 years, 20.5% (22.3%) aged 25 years to 34 years, 18.1% (24.9%) aged 35 years to 44 years, 16.3% (18.5%) aged 45 years to 54 years, 15.5% (14.8%) aged 55 years to 64 years, and 17.4% (4.3%) aged 65 years and over. Of the female population (and survey respondents, in brackets), 11.0% (18.5%) were aged 18 years to 24 years, 18.4% (22.6%) aged 25 years to 34 years, 16.4% (23.5%) aged 35 years to 44 years, 15.0% (17.3%) aged 45 years to 54 years, 18.5% (14.1%) aged 55 years to 64 years, and 20.6% (3.9%) aged 65 years and over. Conclusions For this trial, using a market research company to distribute community surveys resulted in respondents that are broadly representative of the geographic areas where the trial will be conducted, except for individuals over the age of 65 years and older. This finding provides reassurance to investigators and institutional review boards that the views expressed by respondents are representative of the population where the trial will be conducted. Level of evidence III

Objective The purpose of this study was to determine the cumulative incidence of diagnosis switching, drug‐free remission, and initiation of a biologic or targeted synthetic disease‐modifying antirheumatic drug (b/tsDMARD) in individuals with seronegative rheumatoid arthritis (RA). Methods Adult residents of Olmsted County, Minnesota, with incident seronegative (rheumatoid factor negative/anti–cyclic citrullinated peptide antibody negative) RA meeting the 1987 and/or 2010 American College of Rheumatology classification criteria were included. Data were collected from January 1, 2005, to December 31, 2023, through manual chart review. Drug‐free remission was defined as a period of at least six months where the individual was no longer taking treatment for RA and did not have evidence of active inflammatory arthritis upon evaluation by a rheumatologist. We calculated the 10‐year cumulative incidence of a change in diagnosis, adjusting for the competing risk of death, and of drug‐free remission and initiation of a b/tsDMARD, adjusting for the competing risks of death or change in diagnosis. Results A total of 176 individuals with seronegative RA (68% women) were included. The 10‐year cumulative incidence of a change in diagnosis was 12.8% (95% confidence interval [CI] 8.7%–18.9%). The most common change in diagnosis was to spondyloarthritis (4%). The 10‐year cumulative incidence of drug‐free remission and initiation of a b/tsDMARD was 26.6% (95% CI 20.7%–34.2%) and 19.9% (95% CI 14.7%–26.9%), respectively. Over a median follow‐up of 11.8 years, 49 individuals entered drug‐free remission. Conclusion After initial diagnosis of seronegative RA, about 13% of individuals had a change in diagnosis and a quarter experienced drug‐free remission within 10 years.

Abstract An 86-year-old woman with metastatic non-small cell lung carcinoma on Pembrolizumab presented to the Emergency Department with diabetic ketoacidosis; after appropriate treatment she was transitioned to basal/bolus insulin regimen. She had chosen to receive Immune Checkpoint Inhibitor (ICI) instead of radiotherapy, as she wished to preserve her quality of life. With the demands of management of ICI-induced irreversible insulin deficient diabetes she now has substantial worsening of quality of life with considerable anxiety and depressed mood. ICI-induced diabetes and DKA is more common in patients treated with Pembrolizumab than suggested by early trials. In addition to highlighting the importance of counseling patients on the signs and symptoms of hyperglycemia for earlier detection, our case calls for considering the psychological impact of managing a chronic illness in patient education prior to initiating ICI therapy.

Abstract BACKGROUND Children exposed to poverty during frontline therapy for B-lymphoblastic leukemia (B-ALL) experience inferior overall survival (OS), higher relapse rates, and earlier relapse than their peers. Real-world data describing treatment patterns and survival after relapse of B-ALL are lacking and, as a result, the impact of poverty following relapse remains poorly understood. This study evaluated the association of poverty exposure, proxied by insurance status, with post-relapse survival and explored potential explanatory mechanisms, including disease biology and therapy utilization. METHODS Data from Retrospective Study of Contemporary Approaches to First Relapse in B-ALL (ReCALL-1), a multicenter retrospective cohort study of patients < 30 years old with first relapse of B-ALL from 2018-2022, were used. The primary exposure was poverty, proxied by insurance status at relapse, categorized as any private insurance (poverty unexposed), public-only insurance (poverty exposed), no insurance, or unknown; the latter two were not included in comparative analyses due to low numbers. The primary outcome was OS. Secondary outcomes included measurable residual disease (MRD)-negative second complete remission (CR2), relapse after CR2, non-relapse mortality (NRM), with relapse treated as a competing risk, and receipt of cellular and immunotherapies. RESULTS Among 462 patients from 31 U.S. institutions, 41% had private insurance, 47% public-only insurance, 2% no insurance, and 10% unknown. Compared to privately insured patients, those with public insurance were more likely to be Hispanic (56% vs 19%, p<0.001) and have non-English preferred language (30% vs 6%, p<0.001). Non-Hispanic Black patients comprised 9% of those with public insurance and 5% of those with private insurance. Disease characteristics that differed by insurance status at relapse included: Ph-like cytogenetics, more common in publicly insured (23% vs 11%, p=0.002), and infant ALL, more common in privately insured patients (10% vs 5%, p=0.06). Publicly insured patients were more likely to have experienced major organ toxicity during frontline therapy (32% vs 18%, p=0.002) and very early relapse (<18m from diagnosis: 43% vs 32%, p=0.07). Other demographic and disease characteristics, including relapse site and cytomolecular features, were similar by insurance status. With a median follow-up of 51 months from relapse, 4-year OS differed by insurance (log-rank p=0.02): 72% for private vs 56% for public insurance. After adjusting for race/ethnicity and language, public insurance increased the hazard of death by 65% (aHR 1.65 95% CI 1.13-2.42, p=0.004). The association remained, but was attenuated, when unbalanced disease characteristics (time to relapse, infant ALL, Ph-like cytogenetics, prior organ dysfunction) were included (aHR 1.47, 95% CI 1.00-2.16, p=0.05). Rates of MRD-negative CR2 were similar (85% public vs 87% private). However, point estimates suggested publicly insured patients had more relapse after CR2 (44% vs 37%, p=0.42) and higher 4-year NRM (16% vs 10%, Gray's test p=0.12), though neither were significant. Treatment choices for first relapse did not differ by insurance status. Utilization of intensive reinduction (91% vs 95% for public vs private, p=0.18) and clinical trial enrollment (22% for both) were similar. Receipt of cellular therapies did not significantly differ by public vs private insurance: Chimeric Antigen Receptor-T cells (CAR-T: 38% vs 34%, p=0.41), hematopoietic cell transplant (HCT: 42% vs 47%, p=0.32). Use of blinatumomab trended higher in those with private insurance (36% vs 45%, p=0.07), and inotuzumab was similar (11% for both, p=0.87). Median time from relapse to CAR-T or HCT was comparable (to CAR-T 102 vs 100 days, to HCT 117 vs 114 days). CONCLUSION This is the first study to demonstrate differential survival by insurance type, a proxy for poverty exposure, after first relapse of B-ALL. In this large multicenter, real-world cohort, patients with public insurance had worse OS after relapse than those with private insurance. The survival disparity existed independent of other SDOH factors. Adjustment for known high-risk disease features only partially accounted for the observed differences in OS. In contrast, rates of MRD-negative CR2 and receipt of CAR-T and HCT were comparable across insurance types. These findings highlight the urgent need to identify effective interventions that will ensure equitable outcomes after relapse.

Abstract Chimeric antigen receptor T (CART) cell therapy has shown unprecedented success in the treatment of hematological malignancies. However, the immunosuppressive tumor microenvironment of both solid and liquid tumors is a major barrier for CART efficacy. Immunosuppressive myeloid cells inhibit CART efficacy. Colony-stimulating factor 1 receptor (CSF1R) promotes macrophage proliferation, survival, and anti-inflammatory response and is upregulated on myeloid cells in many cancers, making CSF1R an appealing therapeutic target. We aimed to test the interactions between CSF1R, myeloid cells, and CART response, and to study CSF1R targeting in the context of CART cell therapy. We utilized CD19-targeted CART cell therapy (CART19) in lymphoma preclinical models and thyroid-stimulating hormone receptor (TSHR)-targeting CART cell therapy (TSHR-CART) in thyroid cancer models. We first measured CSF1R expression on myeloid cells in vitro. Suppressive myeloid cells, polarized from healthy donor monocytes using an anaplastic thyroid cancer (ATC) cell line, 8505c, or mantle cell lymphoma cell line, JeKo-1, significantly upregulate CSF1R (p=0.0002) compared to media control. After polarization, myeloid cells significantly inhibit TSHR-CART and CART19 antigen-specific proliferation compared to freshly isolated monocytes (p=0.012, p<0.01, respectively) indicating that suppressive myeloid cells contribute to CART failure in multiple tumor types. We then performed single cell RNA sequencing on peripheral blood taken at baseline from patients with mantle cell lymphoma treated with CART19. We found a significant increase of CSF1R on monocyte subsets in CART19 non-responders vs responders (padj=0.0037). Next, we determined the impact of CSF1R blockade on CART cell therapy in vivo. Using lymphoma models, B6 mice were engrafted with the luciferase+ CD19+ murine lymphoma cell line TBL-12 (1x106 cells i.v. 1 day after 250 mg/kg cyclophosphamide). Tumor burden was significantly reduced after 7 days of treatment with low dose CART19 (0.5x106 i.v.) + anti-mouse CSF1R antibody (16mg/kg i.p. twice weekly) vs CART19 + IgG control (p=0.0146). We then tested the impact of CSF1R blockade in mice experiencing tumor relapse. After TBL-12 engraftment, mice were treated with anti-mouse CSF1R or IgG control antibody followed two days later with high dose CART19 (1x106 i.v.) to induce remission. Mice were re-challenged with TBL-12 14 days later. Mice treated with anti-mouse CSF1R antibody showed increased tumor control and significantly higher survival (p=0.0416). Using thyroid cancer models, first we identified high macrophage infiltration in biopsy specimens of patients with ATC compared to normal thyroid tissue and benign thyroid tumors through immunohistochemistry (IHC) of patient samples (p<0.01, p<0.0001, respectively). To test if macrophages are associated with CART failure in ATC, NSG mice were subcutaneously engrafted with 1x106 TSHR+ THJ529 tumors, an ATC cell line derived from patient biopsy samples from the Mayo Clinic thyroid cancer biobank, followed by i.v. treatment with 1x107 TSHR-CART or untransduced T cell control (UTD). IHC staining of tumor tissue collected at experimental endpoints revealed a dose-dependent, significant infiltration of human CD3+ T cells into the tumors which inversely correlated with murine F4/80+ myeloid cell and murine YM-1+ M2 macrophage infiltration. This led us to hypothesize that myeloid cell targeting with CSF1R blockade can improve TSHR-CART efficacy. We utilized the same ATC model described above to test CSF1R targeting. When TSHR+ THJ529 tumors reached approximately 100 mm3, mice were randomized by tumor volume and treated with CSF1R inhibitor PLX5622 (600mg/kg) or vehicle control, followed by either UTD or TSHR-CART cells 5x106 i.v. Mice treated with TSHR-CART + PLX5622 showed significantly improved tumor control vs TSHR-CART + vehicle control (p<0.0001) or UTD + PLX5622 (p<0.0001). Together, our results highlight immunosuppressive myeloid cells which express CSF1R as a major cell type limiting CART efficacy in both solid and liquid tumors. We also identified a strategy to overcome myeloid-mediated suppression of CART cells by targeting CSF1R with antibody or small molecule inhibitors.

Abstract Chimeric antigen receptor T (CART) cell therapy is potentially curative in certain hematological malignancies, resulting in several FDA approvals since 2017. However, widespread adoption is limited by 1) cost and complexity of an autologous platform, 2) severe toxicities, and 3) limited durable remissions. To better predict CART efficacy and toxicity, live CART imaging would allow the realtime assessment of in vivo CART expansion and trafficking. Incorporation of the sodium iodide symporter (NIS)in CART cells (NIS+ CART cells) allows the CART cells to be tracked via PET imaging at high sensitivity. PET signal correlates with CART expansion and toxicity in mouse models (PMID: 34244299). Building on this platform, we aimed to track xenogeneic CART cells to monitor for their rejection, utilizing dogs with spontaneous lymphoma. Canine diffuse large B-cell lymphoma (DLBCL) affects over 250,000 dogs a year and has shared pathologic, biologic, phenotypic, genetic, immunotherapeutic targets, and treatment responses with human DLBCL, providing strong rationale for studying canine lymphoma as a pre-clinical model. We therefore initiated a USDA-CVB regulated pilot study and treated companion dogs with naïve canine CD20 (cCD20)+ DLBCL with human-derived TRAC- and B2M-KO NIS+ CART cells which targeted cCD20. First, we tested and optimized a chemotherapy regimen to achieve optimal lymphodepletion in dogs. Healthy beagles were treated with pentostatin (pen) on days 2-4 (0.125, 0.25, or 0.5 mg/kg i.v.), cyclophosphamide (cy) on day 4 (400 mg/m2 i.v.), or combination cy/pen. The most robust decrease in lymphocytes was seen with combination cy/pen (all cy/pen doses vs all other treatments, p≤0.0001) and in neutrophils with cy or cy/pen (all cy/pen doses vs all pen doses, p≤0.0001; cy vs 0.25 mg/kg pen, p≤0.001; cy vs 0.5 mg/kg pen, p≤0.0001). All lymphodepletion was transient and recovered within two weeks. All regimens were well-tolerated, and we therefore used 0.5 mg/kg pen plus 300 mg/m2 cy for the companion dog pilot study. Next, we assessed the feasibility of generating cCD20-targeted NIS+ human CART cells (CART20-NIS) with double KO (DKO) of TRAC and B2M. Primary T cells from healthy human donors were transduced with bicistronic lentivirus encoding 41BB-costimulated cCD20-targeted CAR and NIS. Two days after transduction, cells were transfected with CRISPR Cas9-gRNA targeted to TRAC and B2M. The resulting CART20-NIS DKO cells were expanded until day 11 and then were further depleted for CD3 and cryopreserved. CART20-NIS DKO cells demonstrated high CAR expression (untransduced T cells (UTD) vs CART20-NIS DKO, p<0.0001) and KO efficiency (TCRαβ and HLA-ABC expression of UTD vs CART20-NIS DKO, p<0.0001). NIS functionality was confirmed via a qualitative in vitro I-125radioactive uptake assay. We then assessed antigen specificity and activity in vitro. CART20-NIS DKO or UTD were co-cultured with either wildtype or cCD20-overexpressing K562 cells for three days. Target cell killing was then quantified via flow cytometry. CART20-NIS DKO cells demonstrated significant cytotoxicity against cCD20+ K562 cells (UTD vs CART20-NIS DKO, p<0.0001). Lastly, we treated two companion dogs in a USDA-CVB regulated pilot study of CART20-NIS DKO cells in naïve cCD20+ DLBCL. Following lymphodepletion on days -5 to -3, on day 0, dogs were i.v. administered 1 x 108 CART20-NIS DKO cells and intratumorally administered 2.5 x 107 CART20-NIS DKO cells in two lymph nodes, with contralateral saline injection as controls. Dogs underwent 18F-TFB-PET imaging on days 1, 3, and 7, which showed evidence of CART20-NIS DKO cells both in the CART-injected lymph nodes and distal lymph nodes, as determined by SUVmax. Both dogs sustained clinical remission for the duration of the 21-day study, as determined by sum of longest diameters and accompanying x-ray radiology and abdominal ultrasound. Histology showed marked reduction of CD20 cells in the lymph nodes. Organ and hematological toxicities were minimal, and dogs remained in remission for 95 and 101 days post-CART. In summary, this study demonstrated that xenogeneic CART cells were well-tolerated, induced remission, and were visualized via PET imaging in companion dogs with lymphoma. These results are promising both for the treatment of companion dogs with novel cell therapies and for the translation of novel CART cell therapies into human clinical trials.

Abstract Background: Despite recent therapeutic advances in AS cHL, PET-adaptive chemotherapy-based regimens remain a first-line treatment option, especially for patients (pts) without access to novel agents. Most PET-adaptive regiments start with either 2 cycles of escBEACOPP (escBEACOPP2) or ABVD (ABVD2) prior to interim PET assessment (iPET), followed by treatment de-escalation or intensification by iPET result. There have been no direct comparisons of escBEACOPP2 vs ABVD2 PET-adaptive regimens. Additionally, the AS-Hodgkin Lymphoma International Prognostication Index (A-HIPI) score (Rodday JCO 2023) has not been fully explored in escBEACOPP2 regimens. Therefore, we utilized multiple analytic approaches to compare outcomes of PET-adaptive escBEACOPP2 and ABVD2 regimens, while adjusting for and assessing the effect of the baseline A-HIPI score. Methods: Through the global HoLISTIC Consortium (www.hodgkinconsortium.com), we obtained individual patient data from 4 AS clinical trials of newly diagnosed cHL pts treated with PET-adaptive escBEACOPP2 (AHL2011) or ABVD2 (SWOG0816, RATHL, HD0607) regimens. Pts were restricted to age 18 to 65 years (y), stage IIB-IV, and PET-adapted treatment arms. Positive iPET was based on Deauville score >3. Treatment effects of escBEACOPP2 vs ABVD2 (reference) on 5y progression-free survival (PFS) and overall survival (OS) were first evaluated using Cox models. Importantly, baseline disease risk was adjusted for using the 5y PFS or OS A-HIPI score (including stage, sex, age, bulk, lymphocyte count, albumin, white blood cell count), where higher scores indicate higher predicted risk (scale 1-100). The A-HIPI was modeled per 1 standard deviation (SD) increase. Our multistate model (MSM) comprised 4 health states: diagnosis, sustained remission at 1yr, treatment failure, and death from any cause. Pts all started in the diagnosis state and transitioned to other states without return to prior states. We assessed the impact of treatment on transitions (except binary sustained remission at 1y) that involved ≥5 pts per treatment group, adjusting for A-HIPI for all transitions. All effects are reported as adjusted hazard ratios (aHRs) with 95% confidence intervals (CI). Results: 2,381 AS cHL pts were included (n=372 for escBEACOPP2, n=2009 for ABVD2). For escBEACOPP2 and ABVD2 groups (median follow-up 60 and 56 months), respectively, the mean baseline A-HIPI scores (i.e., predicted 5y PFS event rate) were 25.1 (SD=6.3) and 22.9 (SD=6.5), the rates of positive iPET were 11.3% and 17.0%, 5y PFS was 87.0% and 79.5%, and 5y OS was 96.3% and 94.6%. In Cox models, escBEACOPP2 was associated with significantly improved PFS compared to ABVD2 (aHR=0.54, 95% CI=0.39-0.73); the aHR for OS was 0.67 (95% CI=0.37-1.19). Notably, higher risk A-HIPI score was associated with significantly worse PFS (aHR=1.42, 95% CI=1.31-1.54) and OS (aHR=1.60, 95% CI=1.47-1.76), independent of treatment. In the MSM, the effect of escBEACOPP2 compared to ABVD2 regimens was modeled in 3 transitions with adjustment for A-HIPI score: those treated with escBEACOPP2 had lower likelihood of treatment failure with (aHR=0.38, 95% CI=0.22-0.66) or without (aHR=0.61, 95% CI=0.40-0.92) sustained remission at 1y; the aHR for treatment failure to death was 0.55 (95% CI=0.25-1.22). Higher risk baseline A-HIPI score was associated with higher likelihood of treatment failure and death without sustained remission at 1y (aHR=1.43, 95% CI=1.28-1.61 and aHR=2.50, 95% CI=1.80-3.49, respectively). Furthermore, higher risk baseline A-HIPI score was associated with higher likelihood of treatment failure after achieving sustained remission at 1y (aHR=1.29, 95% CI=1.12-1.48) as well as death after treatment failure (aHR=1.54, 95% CI=1.27-1.86). Conclusions: Comparing newly diagnosed adult AS cHL PET-adaptive regimens with adjustment for baseline A-HIPI score, we found pts treated with escBEACOPP2 on AHL2011 had improved PFS versus pooled pt data from 3 ABVD2 trials. Using MSM, we further demonstrated that escBEACOPP2 was associated with a lower likelihood of treatment failure with or without achieving sustained remission at 1yr. In addition, baseline A-HIPI score was associated with PFS and OS, which was independent of treatment. Finally, higher baseline A-HIPI scores maintained prognostic impact throughout the disease course, including increased risk of treatment failure for pts in remission at 1yr and death after treatment failure.

Abstract Introduction: Chronic lymphocytic leukemia (CLL) is marked by impaired T cell responses that limit the efficacy of immunotherapies. CD19-directed chimeric antigen receptor T cell therapy (CART19) received FDA approval to treat CLL in 2024. However, itsefficacy in CLL patients remains low, with durable remission rate of ~20%. Ibrutinib, a Bruton tyrosine kinase inhibitor, has shown synergy with CART19 in preclinical and early clinical studies. We aimed to study the impact of ibrutinib on CART19 utilizing clinical samples from the pivotal clinical trial E1912 (NCT02048813) of ibrutinib/rituximab (IR) vs fludarabine/cyclophosphamide/rituximab (FCR). This trial led to the FDA approval of first line ibrutinib in CLL. Methods: We generated CART19 incorporating either CD28 (28ζ) or 41BB (BBζ) costimulatory domains from T cells isolated from patients with CLL treated with IR or FCR on the E1912 trial. IR samples were collected at baseline (pretreatment), 6- or 12-months during treatment, or at progression. FCR samples were collected at baseline, 15-18 months during treatment, or at progression. For animal experiments, NOD-SCID-γ-/- (NSG) mice were intravenously (i.v.) engrafted with 1×10⁶ luciferase⁺ CD19⁺ JeKo-1 cells. Upon tumor establishment (~10⁸ photons/sec by bioluminescence imaging), mice were randomized and treated i.v. with 1×10⁶ 28ζ or BBζ derived from longitudinal IR- or FCR-treated patient samples. To further assess the impact of prolonged ibrutinib exposure on CART19 function, we chronically stimulated healthy donor-derived 28ζ or BBζ in vitro for 22 days with JeKo-1 cells and 100nM ibrutinib or vehicle control and then tested CART19 functions. Finally, we performed single cell RNA sequencing (scRNAseq) of longitudinal patient T cell samples treated with IR. Results: Ex vivo T cell expansion was significantly enhanced following IR compared to FCR. BBζ generated from IR-treated patients after 6 months of ibrutinib exhibited enhanced in vitro expansion (p=0.016) and antigen-specific proliferation (p=0.038) in the presence of JeKo-1 cells. This corresponded with significantly improved antitumor efficacy and prolonged survival in vivo compared to cells generated at disease progression (p=0.017). In contrast, 28ζ showed no significant differences in efficacy across IR studied treatment timepoints. BBζ from FCR-treated patients did not show differences in tumor control based on treatment timepoints but demonstrated shorter survival compared to BBζ from IR-treated patients. 28ζ from FCR-treated patients generated at disease progression demonstrated significantly reduced tumor control and shorter survival compared to 28ζ produced at baseline (p=0.0002), indicating detrimental effects of FCR treatment on the therapeutic potential of CART products. To further investigate the impact of prolonged ibrutinib exposure, we utilized in vitro models of CART exhaustion. We found that, after repeated antigen stimulation with JeKo-1 cells, ibrutinib-treated BBζ had significantly increased antigen-specific degranulation (p=0.005), and cytokine production (IL-2, p=0.045; TNF-α, p=0.003; IFN-γ, p=0.001), compared to BBζ treated with vehicle control. In contrast, 28ζ did not show improved degranulation or cytokine production in cocultures with ibrutinib. Prolonged ibrutinib exposure led to downregulation of FOXO3, a transcription factor associated with T cell exhaustion which has been shown to cause dysfunction specifically in BBζ. These results further indicate that ibrutinib may be more beneficial to BBζ. Indeed, scRNAseq revealed that after ≥ 6 months of ibrutinib, T cells showed downregulation of inflammatory, oxidative stress, and apoptosis pathways, paralleled by modest activation of MYC and E2F targets. However, at disease progression, T cells under ibrutinib treatment displayed marked upregulation of interferon signaling, inflammation responses, and stress-related programs, indicating a shift toward immune reactivation or dysfunction.Conclusion: Our study shows that BBζ, but not 28ζ, generated 6 months during ibrutinib treatment has superior antitumor activity and persistence compared to similar later timepoints or FCR-treated counterparts. Prolonged ibrutinib exposure enhances BBζ function by modulating stress and apoptotic pathways. These findings highlight the importance of pre-CART treatment timing and support ibrutinib as a potential enhancer of CART cell fitness in CLL.

Abstract Recent studies have highlighted the important but pleiotropic role of IL-4 in response to chimeric antigen receptor T (CART) cell therapy. IL-4 was identified as a regulator of dysfunction in CD19-targeting CART cells with a CD28 costimulatory domain (CART19-28ζ) through a genome-wide CRISPR knockout screen as well as RNA and ATAC sequencing of baseline and exhausted healthy donor CART19-28ζ cells and pre-infusion CART19-28ζ cell products from responders and non-responders in the ZUMA-1 trial (Stewart et al, Nat Commun., 2024). However, interrogation of a single-cell atlas fromactivated pre-infusion CART cell products from patients with acute lymphoblastic leukemia revealed a beneficial role for IL-4 and other type 2 cytokines in the promotion of long-term response (Bai et al, Nature, 2024). In this report, we aim to further understand how IL-4 can be detrimental to CART cell therapy. To study the regulation of CART19-28ζ cells by IL-4, we performed RNA sequencing on CART19-28ζ cells that were stimulated with CD19+target cells in the presence of either 20 ng/mL hrIL-4 or diluent for 24 hours, one week, or two weeks in an in vitro model for exhaustion as previously described (Stewart et al, Nat Commun., 2024). We observed that hrIL-4 treatment drove the development of a distinct transcriptomic profile after chronic stimulation for one week (up genes = 3253 and down genes = 3645) or two weeks (up genes = 6435 and down genes = 6224) but not following 24 hours of activation (up genes = 0 and down genes = 0). Gene set enrichment analysis on the differentially expressed genes following two weeks of chronic stimulation showed that hrIL-4 treatment pushes CART cells to a more exhausted and less naïve phenotype (FDR = 0.015) and leads to a broad downregulation in cytokine signaling (FDR = 0.012). To investigate transcription factors associated with IL-4-induced CART cell dysfunction, we used BETA analysis with publicly available transcription factor binding sites for STAT6, GATA3, and EOMES and our differential expression data. EOMES (p = 0.039), but not STAT6 (p = 0.999) nor GATA3 (p = 0.999) significantly regulated the differentially expressed genes. In support of these findings, the addition of hrIL-4 also significantly upregulated EOMES expression after one (p < 0.0001) and two (p < 0.0001) weeks of chronic stimulation and downregulated the expression of TBX21 (p < 0.0001), a binding competitor of EOMES, following two weeks of chronic stimulation. To evaluate if EOMES is critical for IL-4-induced CART cell dysfunction, we treated either control or EOMES CRISPR knockdown CART cells with 20ng/mL hrIL-4 during chronic stimulation. EOMES knockdown reduced IL-4-induced CART19-28ζ cell dysfunction as seen by an increase in antigen-specific expansion (p = 0.046) and an increase in cytokine production including IL-2 (p < 0.0001), TNF-α (p = 0.006), and IFN-γ (p = 0.020). Having identified that EOMES is at least partially responsible for IL-4-induced CART cell dysfunction, we evaluated EOMES binding sites in human CART19-28ζ cells with CUT&RUN sequencing. Our analysis revealed EOMES binding at hypersensitivity site 2 (HS2) in the IL-4 locus which, in a previous study, was more accessible in both exhausted healthy donor CART19-28ζ cells and pre-infusion CART cell products from non-responders in the ZUMA-1 clinical trial (Stewart et al, Nat Commun., 2024). To determine how HS2 can impact CART cell functions, we generated CART19-28ζ cells from either wildtype (HS2+/+) or HS2-deleted (HS2-/-) murine T-cells and tested them in a syngeneic model of lymphoma generated by engrafting C57BL6 mice with luciferase+ TBL12 cells (1x106, tail vein injection on Day -3) following preconditioning (250mg/kg cyclophosphamide, intraperitoneal injection on Day -4). Following randomization of mice via tumor burden (n = 5 per group), mice were treated with either HS2+/+ or HS2-/- CART19-28ζ cells (1x106 cells, tail vein injection). In this model, HS2-/- CART cells showed enhanced expansion (p = 0.022), increased anti-tumor activity (p = 0.018), and improved overall survival (p = 0.003). Together, our study shows that IL-4 induces a distinct cellular fate in CART19-28ζ cells in the presence of chronic antigen-specific stimulation at least partially through the upregulation of EOMES. Further, the epigenetic regulation of CART cells by the IL-4 enhancer HS2 can significantly influence CART cell activity, and deletion of HS2 can improve CART cell therapy.