About

Marco Ruella, MD, is an Associate Professor of Medicine (Hematology-Oncology) at the University of Pennsylvania’s Perelman School of Medicine. He specializes in treating patients affected by hematological cancers, particularly lymphoma, myeloma, and leukemia, with a focus on immunotherapy approaches. His laboratory research concentrates on understanding the mechanisms of relapse after chimeric antigen receptor T cell (CAR T) immunotherapies, aiming to design innovative combined immunotherapies for relapsing or refractory leukemia and lymphoma. Dr. Ruella obtained his medical degree with high honors from the University of Torino, Italy, where he also completed his specialization in clinical hematology. He has held positions as an attending physician at Mauriziano Hospital and as an instructor at the University of Torino's Biotechnology School. His postdoctoral training and early academic career took place at the University of Pennsylvania, where he was a Post-doctoral Fellow and Instructor at the Center for Cellular Immunotherapies. He served as Associate Director of Dr. Carl H. June’s laboratory before being appointed Assistant Professor of Medicine in 2018, and later obtaining tenure as an Associate Professor in 2025. Throughout his career, Dr. Ruella has been recognized with numerous awards, including fellowships, research scholar awards, and grants from prominent organizations such as the NIH, AACR, and ASH. He serves as Senior Editor for Molecular Cancer Therapeutics and Associate Editor for the Journal of Immunotherapy of Cancer. He is also the inaugural Chair of the SITC Cellular Therapy Committee and the immediate past-Chair of the ASH Scientific Committee on Transplantation Biology and Cellular Therapy. Clinically, he treats patients with hematological cancers at the Hospital of the University of Pennsylvania, integrating his research insights into patient care. Additionally, he is involved in biotech industry advisory roles and founded viTToria Biotherapeutics, a startup developing next-generation CAR T-cell immunotherapies, with its first product currently in clinical evaluation.

Research topics

• Cancer research
• Internal medicine
• Immunology
• Oncology
• Medicine

Selected publications

• SPTBN2 promotes an immunosuppressive tumor microenvironment and cross-resistance to anti-cancer therapies

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-04-01

  articleOpen access

  Immunosuppressive tumor microenvironment (TME) inactivates CD8+ cytotoxic lymphocytes (CTLs). Here, we identify SPTBN2 spectrin as a key immunosuppressive regulator induced in CTLs in response to nutritional deficit. In human pancreatic and colorectal cancers, SPTBN2 expression negatively correlated with CTL infiltration and patients' survival. In TME of mouse pancreatic and colorectal adenocarcinomas, SPTBN2 inactivated intratumoral CTLs, stimulated tumor growth and conferred cross-resistance to anti-cancer therapies. SPTBN2 knockout protected CAR T-cells from trogocytosis and increased their memory state. SPTBN2 maintained levels of cell surface proteins such as BTLA that undermine CAR T-cell cytotoxicity and promote exhaustion. Re-expression of BTLA largely reversed phenotypes in SPTBN2-deficient CAR T-cells. In manufactured CAR T cells, SPTBN2 was associated with their clinical failure in pediatric patients with leukemia. Accordingly, ablation of SPTBN2 in CAR T-cells increased their cytotoxicity, in vivo persistence and therapeutic effects indicating that SPTBN2 can be targeted to increase the efficacy of anti-cancer therapies.

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• Epigenetic reprogramming via EZH1/2 inhibition enhances T cell-mediated immunotherapies against multiple myeloma

  Blood · 2025-11-03

  articleOpen accessSenior author

  Abstract Introduction: Although BCMA-targeted CAR-T therapies and CD3×BCMA bispecifics (e.g., teclistamab) have shown clinical promise, most r/r MM patients eventually relapse due to tumor-intrinsic resistance and T-cell dysfunction. Emerging evidence implicates epigenetic regulators promote immune evasion, tumor cell stemness, and therapeutic resistance in MM. We previously demonstrated that the catalytic subunits of the polycomb repressive complex 2, EZH1 and EZH2, contribute to immune escape and resistance to immunotherapies in germinal center–derived B-cell lymphomas, thus influencing the efficacy of anti-CD19 CAR-T therapy. In MM, EZH2 is frequently overexpressed in cytogenetically high-risk disease and at relapse. We, therefore, hypothesized that inhibition of EZH1 and EZH2 could enhance T cell–based immunotherapy for MM by both directly impairing tumor growth and survival, and by boosting T cell–mediated cytotoxicity.Methods and Results: We first demonstrated a strong tumor-intrinsic anti-clonogenic effect of the EZH2 inhibitor (tazemetostat) and the dual EZH1/2 inhibitor (valemetostat) on the MM cell line RPMI-8226. Colony formation assays revealed a significant, progressive reduction in clonogenic growth following treatment with tazemetostat and valemetostat, with the most pronounced effect observed with dual inhibition (day 10, number of colonies, RPMI-8226: DMSO vs EZH1/2i, 622.5 vs 292.5; p=0.0046). We next employed clinically relevant anti-BCMA CAR-T constructs and performed long-term killing assays combining MM cell lines with EZH1/2 inhibitors, with or without CAR-T cells. Notably, EZH1/2 inhibitors significantly enhanced cytotoxicity against MM cell lines (e.g., RPMI-8226; day 5; CAR-T-BCMA+EZH1/2 inhibitor vs CAR-T-BCMA+DMSO, p<0.001). Similarly, in assays using the FDA-approved anti-CD3/anti-BCMA bispecific antibody teclistamab and human T cells, we observed an additive tumor-killing effect (48 hrs; teclistamab+T cells+EZH1/2 inhibitor vs teclistamab+T cells+DMSO, p<0.01). Importantly, EZH1/2 inhibition did not impair T-cell viability or effector function. ELISA of co-culture supernatants showed dose-dependent increases in IL-2 and TNF following teclistamab dose escalation, with no significant impact from the inclusion of EZH1/2 inhibitor, confirming intact T-cell cytokine production (24 hrs; teclistamab 1 nM; IL-2 [pg/mL]: DMSO vs EZH1/2 inhibitor, 29 vs 23.7, p=ns; TNF: 503 vs 528, p=ns). Finally, we developed an intraosseous orthotopic xenograft model of MM using RPMI-8226 cells. Three days after tumor implantation, mice were administered valemetostat (100 mg/kg, orally, once daily for five weeks) or a vehicle control. One week after the RPMI-8226 injection, the mice were randomized to receive CART-BCMA therapy (0.8 × 10⁶ cells per mouse, intravenously). In this model, continuous valemetostat treatment enhanced the tumor-suppressive effect of CART-BCMA, supported T cell expansion in peripheral blood, and was not associated with any notable toxicities such as weight loss or GVHD. Of note, valemetostat/CART-BCMA combination doubled complete response rate (80% vs 40%) and showed increased serum levels of IFN-γ, a key cytotoxic cytokine, indicating heightened T cell activation and cytolytic function. Mechanistically, EZH1/2 inhibition did not alter BCMA expression at either the RNA or protein level, indicating that target antigen expression was preserved. However, RNA-seq analysis of RPMI-8226 and MM1.S cells treated with EZH1/2 inhibitors revealed upregulation of genes involved in cell adhesion (e.g., LAMA1, LAMA2, VCAN, PECAM1), immunogenic signaling (e.g., CXCL9, CXCL10, CXCL14, CD70, TNFSF9), and differentiation (SDC2), along with downregulation of cell cycle–associated genes. These results suggest that EZH1/2 inhibitors reprogram MM cells to a more immunogenic and less proliferative state, enhancing their susceptibility to immune-mediated attack. Conclusions: These findings support a dual mechanism of action EZH1/2 inhibitors directly suppress MM clonogenicity and immune evasion, while concurrently enhancing the function and cytotoxicity of T cell–based immunotherapies. By preserving T-cell activity and reprogramming tumor cells, EZH1/2 inhibition represents a promising strategy to improve the durability and depth of response to both CAR-T and bispecific antibody therapies in MM. These data support the development of clinical trials combining EZH1/2 inhibitors with CART and bispecific antibodies in r/r MM.

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• Naive CD4+ at apheresis and disease control at infusion are associated with improved efficacy in second-line CAR T-cells

  Blood · 2025-11-03

  articleOpen accessSenior author

  Abstract Introduction: Second-line (2L) anti-CD19 CAR T-cell therapy (CART) with axicabtagene ciloleucel (axi) or lisocabtagene maraleucel (liso) is standard-of-care for patients (pts) with large B-cell lymphomas (LBCLs) who relapse within 12 months after frontline treatment. However, determinants of response and toxicity in 2L real-world settings remain poorly defined. Methods: We retrospectively analyzed 64 consecutive LBCLs pts treated at our institution with 2L axi (n=35) or liso (n=29) between 05/2022 and 12/2024. Multiparameter spectral immunophenotyping (Citek) was performed on peripheral blood collected at apheresis and day 7 post-CART. The primary objectives were to compare the two products in a real-world setting and to identify predictors of efficacy and toxicity. Results: Median age at time of CART was 62 years (21-80yrs); 59% had primary refractory LBCL, 17% had an HGBL, 66% had III/IV-stage and 38% had elevated LDH at infusion. Median vein-to-vein time was 42 days (median axi=36 vs liso=43; p=0.03). Bendamustine was used for lymphodepletion in 89% (axi=83% vs liso=96%, p=0.1). Overall and complete response rates (ORR and CR) at day 90 were 66% and 55%.At a median follow-up of 17 months (4-34mo), 12-month PFS and OS rates were 47% and 78%. Efficacy was comparable between axi and liso (12-month PFS: 51% vs 48%, p=0.8; best CR: 51% vs 58%, p=0.6), despite older age (mean 56 vs 66yrs, p<0.01) and higher IPI (≥3 in 28% vs 72%, p<0.01) in the liso cohort.Cytokine release syndrome (CRS) of any grade occurred in 52% of pts (G≥3 = 3%) and neurotoxicity (ICANS) in 12% (G≥3 = 3%). No non-relapse mortality or secondary malignancies occurred. Axi was associated with higher toxicity, with increased rates of CRS (any grade: 80% vs 17%, p<0.01) and a trend towards higher ICANS (any grade: 20% vs 3%, p=0.06). Due to few severe CRS/ICANS events, comparisons between axi and liso were not performed.Disease reassessment after bridging therapy was available for 92% of pts and 34% were infused in progression. Pts infused with progressive disease had worse outcomes as compared to those with disease control: CR as best response in 15% vs 73% (OR=0.06 95%CI=0.01-0.3, p<0.01), 12-month OS 48% vs 91% (p<0.01), and PFS 21% vs 59% (p<0.01). Bulky disease, extranodal involvement, and elevated LDH at infusion were negative predictors of durable response (defined as PFS≥12 months); interestingly these features were not prognostic when analyzed at the relapse after the frontline therapy.Given the prognostic value of clinical variables at infusion but not at relapse, we explored flow cytometry to identify early predictors of treatment response. Higher CD3⁺ and CD4⁺ T-cell counts at apheresis predicted PFS and CR to CART. Optimal cutoffs (401 CD3⁺/µL, 201 CD4⁺/µL) stratified pts with significantly prolonged 12-month PFS rates (59% vs 29% for CD4⁺; p<0.01). High Naive CD4⁺ T-cell at apheresis strongly correlated with durable responses and prolonged PFS (12-month PFS rates: 12% vs 87%, p<0.01).At day 7, responders showed a trend toward greater CART expansion (CAR⁺/CD3⁺: 17% vs 6%, p=0.1) and higher frequencies of naive CD8⁺CART. In contrast, pts infused with progressive disease had numerically lower CART expansion and a more differentiated effector memory phenotype (CAR+/CD3+: 5% vs 14%, p=0.1 and CD45RA-/CCR7-: 54% vs 38%, p=0.3). CAR⁻ bystander T-cells showed a more naive CD4⁺ and less effector CD8⁺ profile indicating functional divergence from CART.In multivariate analysis including CD4⁺ count at apheresis, disease progression at infusion, and primary refractory status, higher CD4⁺ count remained independently associated with higher odds of CR (OR=4.31, 95%CI=1.15-18.8, p=0.03). Progressive disease at infusion (OR=0.06, 95%CI=0.01-0.3, p<0.01) and primary refractory disease(OR=0.2, 95%CI=0.05-0.8, p=0.03) were associated with lower odds of CR. Conclusions: This is the first real-world study to define clinical and immune correlates of efficacy in 2L CART therapy for LBCL. Both axi and liso are safe and effective. Naïve CD4⁺ T-cell abundance at apheresis predicts long-term outcomes and may serve as a biomarker of T-cell fitness and CART potency. Progressive disease at the time of infusion is associated with inferior response rates and survival outcomes, which may be due to less favorable expansion kinetics and T-cell phenotypes. These findings support immune profiling at apheresis and rational bridging to improve outcomes in early-line CART therapy.

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• Frontline experience with second generation covalent Bruton tyrosine kinase inhibitors for mantle cell lymphoma: A single center experience

  Blood · 2025-11-03

  article

  Abstract Introduction: Mantle cell lymphoma (MCL) is usually an incurable lymphoma with no standard frontline therapy. Increasingly, Bruton tyrosine kinase inhibitors are utilized in frontline therapy, especially in older patients, but data remain limited in younger patients. We report our experience with a cohort of patients with MCL who received frontline second-generation BTKi +/- rituximab. Methods: We reviewed all patients treated at our institution with either acalabrutinib or zanubrutinib (BTKi) +/- maintenance rituximab who had therapy initiated by July 10, 2024. Adverse events (AEs) were graded based on CTCAE v5. We defined patients as either younger or older: patients <65 years old were considered younger unless noted to be autologous hematopoietic stem cell transplant (ASCT) ineligible. Patients ≥70 years were considered older. Patients aged 65-70 were assessed for ASCT eligibility; ASCT-ineligible patients were considered older. A cohort of patients who had frontline standard-of-care chemotherapy (R-HyperCVAD or R-CHOP/R-DHAP) was also collected for comparison to the younger BTKi-treated cohort. Results: Thirty patients received frontline second-generation BTKi. Eleven (37%) were younger and 19 (63%) were older. Twenty-three patients (77%) received acalabrutinib (10 with rituximab maintenance) and 7 patients (23%) received zanubrutinib (4 with rituximab maintenance). Twenty (67%) were male, 27 (90%) were white, and 3 (10%) were black. Median age was 70.9 years (range 44.8-93.4). Twenty-five (83%) had ECOG performance status (PS) 0-1. Twenty-seven patients had advanced stage disease (90%). MIPIb was high risk in 21 (70%); Ki-67 was ≥ 50% in 6/26 (23%) and ≥ 30% in 12/26 (46%). Three (10%) patients had blastoid MCL. Five of 26 (19%) patients had a TP53 aberration. Between younger and older cohorts, there were no significant differences in sex, ECOG PS, blastoid MCL, or presence of TP53 aberrations. MIPIb was significantly higher in older patients (6.3 vs. 7.2, p=0.003). Overall, 24 patients (80%) had an AE related to BTKi, with 7 (23%) experiencing a serious AE (SAE). The most common AEs were bleeding/bruising (33%), infections (n=8, 27%; 1 URI, 4 pneumonia, 1 bacteremia, 1 urinary tract infection, 1 cellulitis; 3 were SAEs), and rash (20.0%). There was no significant difference between rate of any AE or SAE between older and younger patients. Five patients discontinued BTKi due to toxicity (recurrent neutropenia, rash, cellulitis, pneumonitis, dysgeusia) and one due to patient preference. Thirty-two patients were included in the frontline chemotherapy cohort. Twenty-five patients (78.1%) were male, 29 (90.6%) were white, two (6.3%) were black, and one (3.1%) was Hispanic. Median age was 60.3 years (range 27.3-74.5 years). Twenty-eight (87.5%) patients had an ECOG PS 0-1. Median stage was 4 (range 2-4) and median MIPIb was 6.8 (range 5.4-9.6). Nineteen (59.4%) of patients had classical MCL and 13 (40.6%) had blastoid/pleomorphic MCL. Five of 28 (18%) patients had a TP53 aberration. Comparing younger patients who received frontline BTKi and younger patients who received frontline chemotherapy, there were no significant differences in sex, ECOG PS, MIPIb, or presence of TP53 aberrations or proportion of patients who received maintenance rituximab; however, the younger BTKi cohort did have a significantly lower incidence of blastoid MCL (10% vs. 37%, p=0.02). Median follow-up for the entire cohort (n=62) was 58 months; estimated 3-year PFS for the BTKi (n=30) and chemotherapy cohorts (n=32) was 57% (95%CI 34-74%) vs. 43% (95%CI 26-60%). Median follow-up for the younger BTKi and younger chemotherapy cohorts was 35 and 102 months, respectively. Estimated 3-year PFS for the younger BTKi cohort (n=11) vs. younger chemotherapy cohort (n=27) was 52% (95%CI 20-77%) vs. 48% (95%CI 25-61%). Conclusions: Second-generation BTKi with and without rituximab appear safe and effective frontline therapy for MCL regardless of patient age. We also observed excellent BTKi outcomes in younger patients comparable to standard-of-care chemotherapy. Limitations include a higher proportion of patients in the frontline chemotherapy group with blastoid MCL as well as relatively short follow-up in the BTKi cohort. Nevertheless, second generation BTKi may represent an effective frontline therapeutic approach for patients with MCL. Further studies are needed to determine the role of chemotherapy with BTKi vs chemotherapy-free regimens in this setting.

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• Rebuilding immunity: Kinetics and clinical impact of immune reconstitution after CAR T-cell therapy in multiple myeloma

  Blood · 2025-11-03

  articleOpen access

  Abstract Introduction: Chimeric antigen receptor T-cell (CART) therapy frequently results in profound B- and T-cell depletion. The dynamics and clinical implications of immune reconstitution (IR) post-CART in multiple myeloma (MM) remain understudied. Methods Single-center retrospective study of 198 MM patients (pts) treated with ide-cel (n=73) or cilta-cel (n=125) between June 2021 and December 2024. Median follow-up was 20.5 months (mo) (ide-cel 30.6 vs cilta-cel 14.1). We analyzed the kinetics of T-cell and immunoglobulin subsets, excluding values after disease progression, and assessed their association with efficacy outcomes, treatment-related toxicities, and infections. Pts who initiated maintenance therapy were censored at the time of maintenance initiation. Kaplan–Meier analyses were used to assess treatment response and survival outcomes. Results This heavily pretreated cohort included 73.7% and 24.7% of pts with triple-refractory and penta-refractory MM, respectively; 18.7% had high-risk cytogenetics. Lymphocyte expansion kinetics differed based on product. In ide-cel-pts the absolute lymphocyte count (ALC) peaked at Day (D) 7 (300 vs 100 cells/μL), whereas cilta-cel peaked at D12 (450 vs 1100 cells/μL) and demonstrated a delayed but greater sustained expansion from D10 to D24 (AUC ide-cel 7,100 vs cilta-cel 11,400; p<0.01; bootstrap 95% CI [–7.6, –1.6]) despite comparable CD4⁺ and CD8⁺ T-cell proportions at apheresis between products (p>0.05). Among ide-cel–pts, a higher CD4⁺ proportion at apheresis trended toward longer PFS, exceeding the product-specific median (p=0.08), while no such association was observed in the cilta-cel group (p>0.5). Profound immunodeficiency at Month (M) 1, defined as CD4⁺ T-cell counts <50/μL, was associated with a significantly shorter PFS (3.2 vs 22.4 mo, p<0.0001). Median CD4⁺ T-cell count recovery (>150 cells/μL) occurred by M6, coinciding with the median time of Trimethoprim/Sulfamethoxazole exposure (5.6 mo [IQR 1.6–10.7]). To assess humoral immune reconstitution, IgM levels were analyzed, excluding pts with IgM-secreting myeloma and censoring those at time of progression. Ide-cel was associated with earlier immunoglobulin recovery (median IgM≠0), typically by M6 (Median IgM: ide-cel 13 mg/dl [IQR: 0-30.5] vs cilta-cel 0 [IQR: 0-19.5], p=0.06), compared to Year (Y) 1 (Median IgM: ide-cel 37 mg/dl [IQR: 21-56] vs cilta-cel 17.5 [IQR: 0-46.8], p=0.02). IgM levels were also significantly higher in ide-cel recipients at M3 (p<0.001). Immune reconstitution was associated with non-M-Spike bands in 20% of pts (12/61 with detectable IgM at M6). Among ide-cel–pts, higher detectable IgM levels at M3 were associated with inferior PFS (median IgM M3: 28 [PFS≤12 mo], 0 [PFS>12 mo], p=0.004). This association was not observed in cilta-cel-pts, likely related to immature follow-up. The infection rate in the cohort was 68.2% (135/198), with 54% resulting in hospitalizations. Median time to first infection was 1.9 mo [IQR: 0.6-4] with a trend toward earlier infection in cilta-cel (1.8 vs 2.5 mo, p=0.06). Pts with undetectable IgM at M3 who did not receive IVIG had a numerically higher risk of infection (72% vs 30%, p=0.15). These findings link prolonged immunoparesis to increased infection risk, supporting the use of IVIG. Indeed, 82.7% (158/191) of pts received IVIG, with a median initiation time of 1.8 mo post-CART. Among them, 47% discontinued IVIG by a median of 9.3 mo. Pts who received IVIG without evidence of immune reconstitution had similar infection and hospitalization rates as those with immune reconstitution (p>0.05 for each comparison). Conclusion: Immune profiling after CART therapy offers opportunities for risk stratification and intervention. A higher CD4⁺ T-cell proportion at apheresis may serve as a predictive biomarker for improved outcomes in ide-cel recipients, informing product selection or apheresis optimization strategies. Early CD4⁺ lymphopenia and rapid IgM recovery were associated with inferior PFS, suggesting that immune profiling at one month may help identify high-risk pts who could benefit from closer monitoring or early intervention. Cilta-cel induces delayed but more durable expansion, contributing to prolonged immunoparesis. The high incidence of infections, particularly among pts with persistent immune deficits and absent IgM, highlights the need for careful monitoring and infectious prophylaxis ie. use of IVIG to mitigate infection-related morbidity.

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• Data from DLBCL Cells Emerge after CD19 CAR T Cells with Cross-Antigen Resistance and a Gene Signature Predictive of Clinical CAR T-cell Response

  2025-11-03

  articleOpen access

  <div>Abstract<p>Current understanding of lymphoma cell-intrinsic mechanisms of relapse following chimeric antigen receptor (CAR) T-cell treatment of diffuse large B-cell lymphoma (DLBCL) include antigen loss and apoptosis resistance. Herein, CD19 CAR T-cell response and resistance were modeled, and it was identified that treatment-naïve CD19 expression does not correlate with CAR T-cell sensitivity, but resistance is frequently accompanied by reversible downregulation of CD19 that once restored is not paralleled with restored sensitivity to CAR T cell–mediated killing. Profiling a suite of DLBCL cell lines to CD19 CAR T-cell sensitivity reveals that DLBCL cells become nonresponsive to CAR T cell–killing, including to alternative antigen targeting of CD20 or CD22. Leveraging these resistant models, we identified gene signatures present in the CAR T cell–resistant DLBCL cell lines that correlate with patient response to CTL019 in two independent clinical trials. Finally, we show that combination strategies to overcome this resistance, including up-front dual-antigen targeting and combined treatment with an Mcl-1 inhibitor, improve CAR T-cell responses.</p>Significance:<p>We demonstrate that DLBCL cells surviving CD19 CAR T-cell treatment develop a resistance phenotype with a “resistance signature” predictive of clinical CAR T-cell response, mediating cross-resistance between CAR T cells targeting different antigens. Our findings suggest that up-front dual-antigen targeting and combination therapies could improve clinical outcomes.</p></div>

  PublisherDOI

• Cardiovascular outcomes of patients transitioned from ibrutinib to an alternate bruton tyrosine kinase inhibitor for hypertension and/or cardiovascular adverse events

  Blood · 2025-11-03

  article

  Abstract Introduction: Bruton tyrosine kinase inhibitors (BTKi) are highly efficacious oral agents FDA-approved for treatment of specific B-cell malignancies. BTKi are generally administered until disease progression, intolerable toxicity, or death. The emergence of cardiovascular adverse events (CVAE) including hypertension (HTN), arrhythmias, heart failure, and sudden death have limited the use of ibrutinib (Ibr), the first-in-class BTKi FDA-approved in 2013. Hypertension (HTN) is a frequent and cumulative toxicity of Ibr that is associated with increased risk of major adverse cardiac events. Alternate covalent BTKi (acalabrutinib, zanubrutinib) and non-covalent BTKi (pirtobrutinib) have lower rates of CVAE in clinical trials compared with Ibr. The objective of this study was to analyze the real-world incidence of HTN and CVAE in patients (pts) on Ibr and the outcome of pts with new or worsening HTN on Ibr who were then transitioned to an alternate BTKi. Methods: We conducted a retrospective electronic medical record review of pts with hematologic malignancies treated with Ibr from January 2013 to July 2024 at the University of Pennsylvania. Blood pressure (BP), cardiovascular medications, comorbidities, and CVAE were analyzed prior to Ibr (baseline), while on Ibr, and while on subsequent BTKi (acalabrutinib, zanubrutinib, or pirtobrutinib). Eligible pts had at least 3 BP measurements available during each of the following periods: 1) within 12 months of Ibr initiation; 2) while on Ibr; and 3) while on subsequent BTKi. All available BP values were used to calculate medians and means for each therapy period. HTN was defined as elevated systolic BP (SBP) ≥130 and/or diastolic BP (DBP) ≥80 on more than one occasion with physician confirmation of the diagnosis. Worsening HTN was defined as pts with antecedent HTN with an increase in the number or doses of prescribed antihypertensives. Graphpad/R 4.4.0 were used for statistical analysis. Results: A total of 114 pts received Ibr for 408.7 patient-years and 77 (68%) of pts had CLL. The median age was 67 (range 27-86) and 81 (71%) pts were men. On Ibr, 109 (96%) pts had systolic HTN and 100 (88%) had diastolic HTN. Across all BTKi, 74 (65%) pts had a CVAE. CVAE led to Ibr discontinuation in 58 (51.5%) pts, including HTN (n = 20, 18%), atrial fibrillation (n = 25, 22%), other arrhythmia (n = 1, 0.9%), palpitations (n = 2, 1.8%) and hemorrhage (n = 10, 8.8%). Among all pts on Ibr, 67 (59%) had either new onset HTN (n = 49, 43%) or developed worsening HTN with an increase in anti-HTN medications (n = 18, 16%). The median time on Ibr to first elevated SBP and maximum SBP were 32 (95% CI: 24 – 49) and 342 (95% CI: 229 – 604) days, respectively. The median time on Ibr to first elevated DBP and maximum DBP were 114 (95% CI: 83 –199) and 335 (95% CI: 250 – 465) days, respectively. Among the 109 pts with HTN on Ibr, transition to acalabrutinib (n = 67, 61%) or zanubrutinib (n = 33, 20%) resulted in a mean reduction in SBP of -9 mm/Hg (95%CI: -13 to -5.1) and -6 mm/Hg (95%CI: -11 to -0.8), respectively, without a change in number of antihypertensive medications. There were no observed differences in SBP, DBP, or the number of anti-HTN medications among pts with HTN on Ibr who transitioned to pirtobrutinib (n = 9, 8%). Among pts with HTN on Ibr, 45 (41.3%) had resolution of HTN with a median time to resolution (mTTR) of 2,277 days (95% CI: 1,996 – 2,463), although this time estimate is biased by infrequent follow-up. Among 55 pts with documented HTN on Ibr who transitioned to acalabrutinib, 20 (36.4%) had resolution of HTN with mTTR of 1,463 days (95% CI: 976 – NE). Among pts with documented HTN on Ibr who transitioned to zanubrutinib (n = 26) and pirtobrutinib (n = 8), HTN resolved in 8 (30.8%) and 2 (25%) with mTTRs of 683 (95%CI: 606 – NE) and NR (95%CI: 158 – NE) days, respectively. Conclusion: BTKi are associated with increased risk of CVAE; 43% of pts on Ibr developed new onset HTN and 52% of pts discontinued Ibr due to a CVAE. Among pts with HTN on Ibr, transition to alternate covalent BTKi was associated with a reduction in mean SBP as well as resolution of HTN, in some patients, without an increase in number or dose of antihypertensive medications. Despite the development of HTN on Ibr, BP can improve after replacing Ibr with an alternate BTKi.

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• A newly identified role for IL-5 in regulating CART-associated toxicity and efficacy

  Blood · 2025-11-03

  article

  Abstract Background: CD19-directed CART cell (CART19) therapy has transformed the treatment of B-cell malignancies but is limited by life-threatening toxicities, including cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS), as well as low durable remission rates. Our understanding of the pathogenesis of CART-associated toxicities is limited, and although current treatments are effective, refractory cases remain challenging. Methods and Results: To investigate CRS/ICANS pathogenesis and identify new therapeutic targets, we analyzed plasma cytokine kinetics within the first month of CART19 cell therapy in 25 relapsed/refractory (r/r) large B-cell lymphoma (LBCL) and 14 r/r follicular lymphoma (FL) patients (NCT02030834). In this cohort, 22 (88%) patients developed any-grade CRS (73% LBCL, 27% FL) and 4 (16%) developed any-grade ICANS (50% LBCL, 50% FL). Across 30 analyzed cytokines, IL-5 emerged as the second most enriched cytokine based on the median peak-to-baseline (Day 0) ratio in patients who experienced CRS vs. those who did not. The IL-5 ratio was 6.6-fold higher (p<0.05) and 11.9-fold higher (p=0.093) in patients who experienced CRS or ICANS, respectively. Moreover, IL-5 was elevated in patients experiencing CRS on Days 1 and 2 (p<0.05). Prompted by our results, we evaluated the effect of IL-5 neutralization with a monoclonal antibody (mAb) on CART-associated toxicity in a patient-derived B-acute lymphoblastic leukemia (B-ALL) xenograft model. After engraftment (average of 110 human CD19+ cells/µL peripheral blood), NSG mice were randomized based on tumor burden and treated with 3.5×10⁶ CART19 plus either IL-5 mAb or IgG control (10 mg/kg, weekly), or no treatment. Mice receiving IL-5 mAb showed reduced weight loss (p<0.005, Days 13–16) and neuroscore (p<0.05, Day 15), as assessed by a daily, blinded 18-point assessment, vs. the IgG group. Additionally, while CRS-associated cytokines (GM-CSF, IFN-γ, MIP-1β) were significantly elevated in the IgG group (p<0.05) vs. the untreated group, they were not in the IL-5 mAb group. In this model, all mice treated with CART19 cells were able to clear the tumor burden. However, in a follow-up challenge model generated by engrafting NSG mice with 1x106 luciferase+ JeKo-1 cells, mice treated with a combination of 1x106CART19 cells + IL-5 mAb showed reduced weight loss (p<0.0001) and enhanced overall survival (p<0.01) vs. mice treated with CART19 + IgG. Next, we evaluated IL-5 neutralization in an immunocompetent toxicity model. BALB/c mice were treated with 10×10⁶ murine untransduced T cells, CART19 + IL-5 mAb, or CART19 + IgG (10 mg/kg, weekly). Mice receiving IL-5 mAb exhibited significantly reduced weight loss (p<0.05, Day 1) and lower neuroscore (p<0.05, Days 1, 6, and 8) vs. those receiving IgG control, with a consistent trend observed on other days, supporting a protective role for IL-5 blockade in mitigating CART-associated toxicities. To explore the direct effects of IL-5 on CART19 cells, we disrupted either IL-5 (IL-5KO-CART19) or its receptor (IL-5RαKO-CART19) using CRISPR-Cas9. In NSG mice engrafted with 1×10⁶ luciferase+NALM6 cells, mice receiving 1×10⁶ IL-5KO-CART19 vs. IL-5WT-CART19 showed a trend toward reduced weight loss (p=0.08), significantly increased antitumor activity by Day 20 (p<0.05), and improved overall survival (p<0.05), indicating that IL-5 production can significantly impair CART19 cell efficacy. We next evaluated changes in IL-5Rα gRNA representation in a published genome-wide CRISPR screen (PMID: 39266501). CART19 cells with IL-5Rα-targeting gRNAs were enriched following chronic stimulation, indicating a protective effect for IL-5 pathway interruption. In subsequent studies, IL-5RαKO-CART19 cells showed enhanced killing of JeKo-1 cells following 48 hours of co-culture (p<0.05) and reduced co-expression of multiple inhibitory markers (p<0.01) following chronic stimulation with JeKo-1 cells for one week vs. IL-5RαWT-CART19 cells, suggesting an improvement in CART19 cell activity and phenotype following disruption of IL-5Rα. Conclusion: Our data identify IL-5 as a previously unrecognized mediator of CART–associated toxicities and show significant improvement of CART19 activity with disruption of IL-5 signaling either indirectly with an IL-5 neutralizing antibody or directly with CRISPR gene editing. Thus, IL-5 could serve as a dual-purpose target to improve both safety and efficacy of CART cell therapy.

  PublisherDOI

• CCR5 blockade: A therapeutic approach to uncouple CART-BCMA expansion from CART-mediated immune toxicities.

  Blood · 2025-11-03 · 2 citations

  articleOpen accessSenior author

  Abstract Introduction: Chimeric antigen receptor (CAR) T-cell therapy is standard care for relapsed or refractory hematologic malignancies. As clinical use expands, rare but serious toxicities beyond cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are emerging. These include hyperleukocytosis and, specifically, following BCMA-directed CART (CART-BCMA) therapy, delayed neurotoxicity and enterocolitis. We named these post–CART-BCMA complications, distinct from CRS and ICANS, as CART immune-related adverse events (CirAE). CirAE are associated with elevated CART expansion in the first two weeks, suggesting that modulating CART-BCMA proliferation without affecting CART effector function could improve safety and expand therapeutic use. Method: We retrospectively analyzed a cohort of 198 multiple myeloma (MM) patients treated with CART-BCMA (idecabtagene vicleucel [ide-cel] and ciltacabtagene autoleucel [cilta-cel]) at the University of Pennsylvania (June 2021-December 2024) to identify predictors of CirAE and to investigate the mechanisms underlying the early onset of CirAE. Serial serum samples were analyzed using 32-plex proteomics (Luminex). Immune cell subsets were characterized serially by multiparametric flow cytometry. T-cell proliferation was assessed using CellTrace Violet. Cytotoxic CART function was evaluated via luciferase assays. Results: In this large cohort, both peak absolute lymphocyte count (ALC) ≥ 2.4 × 10³/μL and CD4:CD8 ratio >1 at apheresis independently predicted CirAE following CART-BCMA. To investigate mechanisms underlying elevated ALC, we analyzed a unique case (Cilta#1) marked by extreme, polyclonal CART expansion and three distinct post-infusion CirAE: facial palsy, delayed ICANS, and severe enterocolitis. Cilta#1 exhibited hyperleukocytosis (peak ALC: 197.5 × 10³/μL, Day 13) and profound CD4-skewed CART expansion (CD4:CD8 ratio: 12.6). Despite the magnitude, the CART population remained polyclonal, as confirmed by TCR Vβ sequencing and vector integration site analysis (>20,000 unique sites). Whole-exome sequencing of blood and marrow excluded clonal transformation or pathogenic mutations. Longitudinal flow cytometry from pre-lymphodepletion to month 15 showed persistent CD4 skewing, with CART displaying a highly activated, proliferative phenotype(HLA-DR+/Ki-67+). Serum proteomics at day 7, prior to peak expansion, revealed elevated lymphoproliferative cytokines (IL-2, IL-7, IL-15) and chemokines (CCL5, CXCL9, CXCL10). Given these findings, we assessed the dominant proliferative signal via cytokine-stimulated proliferation assays on Cilta#1 CART cells. IL-15 elicited the strongest proliferation and pSTAT5 activation. In vitro, IL-15 induced CCL5 secretion in both Cilta#1 and healthy donor CART (n=3); however, only Cilta#1 cells upregulated CCR5, a pattern absent in donor CART, where CCR5 was actually downregulated after cytokine exposure. These results suggest a potential IL-15–driven CCL5–CCR5 loop sustaining CART expansion in Cilta#1. We hypothesized that this axis contributes to the elevated ALC observed in patients with CirAE. To test this, we cultured healthy donor CART + 25% Cilta#1 day 7 serum, and observed significantly enhanced survival compared to serum from three other cilta-cel patients (p < 0.001). This effect was abrogated by 40 μM maraviroc,an FDA approved CCR5 antagonist, highlighting the key role of CCR5 signaling. Notably, maraviroc suppressed both IL-15–induced and antigen-dependent CART proliferation across multiple donors (n=4), suggesting broader applicability. Importantly, maraviroc did not impair CART viability or anti-myeloma cytotoxicity, as assessed by flow cytometry for CD107a, granzyme B, and MM.1S tumor killing. CCR5 blockade (20 µg/mL) inhibited IL-15–induced proliferation in Cilta#1 CART, further validating CCR5 as a key effector node. Finally, CCR5 knockout impaired antigen-driven proliferation and rendered CART insensitive to maraviroc, confirming on-target specificity.Conclusion: We identified the IL-15–CCL5–CCR5 circuit as a key driver of CART-BCMA proliferation in CirAE and demonstrated that CCR5 blockade safely restrains CART-BCMA expansion while preserving their anti-myeloma activity. These findings support CCR5-directed targeted strategies to selectively modulate CART expansion without compromising efficacy.

  PublisherOA PDFDOI

• Early administration of CD20 x CD3 bispecific antibodies 4-6 weeks after CAR-T infusion for patients with residual or progressive large B cell lymphomas

  Blood · 2025-11-03

  articleOpen access

  Abstract Background: CD19-directed CAR T-cell therapy (CAR-T) has improved outcomes and altered the treatment landscape for patients with relapsed/refractory large B-cell lymphomas (r/r LBCL). Despite these improvements, 60-70% of patients do not have long term remissions after CAR-T. CD20 x CD3 bispecific antibodies (BsAbs), such as mosunetuzumab and glofitamab, have demonstrated efficacy in LBCL relapsing after CAR-T (Chong Blood Advances 2025). We hypothesized that BsAbs could enhance the efficacy of CAR-T by reducing antigen-negative escape and enhancing CAR-T cell activation and persistence. To evaluate this hypothesis, we designed a phase IIa trial of early administration of mosunetuzumab or glofitamab within 31-45 days of CAR T-cell infusion. Methods: This is a multi-center clinical trial of early administration of BsAb for patients with r/r LBCL who receive standard of care CAR-T and have a partial response (PR), stable disease (SD), or progressive disease (PD) at day 30 post CAR-T infusion. BsAb is administered day 31-45 post CAR-T. Patients receive 2 cycles of BsAb (Cohort 1, mosunetuzumab; Cohort 2, glofitamab) and are assessed for response. Patients with complete response (CR) or PD after 2 cycles of BsAb discontinue BsAb; patients with PR or SD are continue BsAb every 3 weeks for up to 1 year and every 24 months during the second year. Efficacy is measured by the CR rate at 24 weeks after initiation of BsAb. CAR-T expansion in blood is assessed by qPCR. Enrollment to Cohort 1 (mosunetuzumab) is complete and Cohort 2 (glofitamab) enrollment is ongoing (NCT04889716). Results: Eight patients, 5 male and 3 female, with a median age of 63 years (range 47-78) were enrolled between January 2022 and May 2025, and included 7 patients with diffuse large B-cell lymphoma NOS (GCB-like [n=4], ABC-like [n=2]) and 1 patient with high grade B-cell lymphoma (double-hit). Patients had a median of 3 prior lines of therapy (range 2-7); 5 patients were primary refractory, 6 patients had extranodal disease, and 5 patients had elevated LDH at CAR-T infusion. Prior CAR-T products included tisagenlecleucel (n-2) and lisocabtagene maraleucel (n=6). The median time from CAR-T cell infusion to BsAb treatment was 42 days (range 33-45). Pre-BsAb responses to CAR-T at Day 30 included 4 PR, 1 SD, 3 PD. Within 30 days after CAR-T infusion and prior to treatment with BsAb, three patients had cytokine release syndrome (CRS) (n=2, grade 1; n=1, grade 2); no ICANS was observed. Mosunetuzumab (n=6) was generally well tolerated; CRS occurred in 3 of the 6 (50%) patients and was low grade (n=2, grade 1; n=1 grade 2). One patient received corticosteroids. One patient had a grade > 3 adverse event related to mosunetuzumab (2 episodes of grade 4 neutropenia, which responded to G-CSF and delay of mosunetuzumab). No CRS occurred in the 2 patients who received glofitamab. No patients developed ICANS. No unexpected adverse events have occurred. The best overall response rate (ORR) in the mosunetuzumab cohort (n=6) was 67% (1 CR, 3 PR, 2 SD, 1 PD). Four patients improved their CAR-T response status after the addition of mosunetuzumab (1 PD to SD, 2 SD/PD to PR, and 1 PR to CR). At 24 weeks, the best ORR was 50% (1 CR, 2 PR, 3 PD). With median follow-up of over 3 years, 1 year progression-free survival is 33% (95%CI 5-68); 1 year duration of response is 50% (95%CI 6-84). Response assessment in the glofitamab cohort is forthcoming. We also assessed changes in T cells and CAR-T cells in both cohorts. After starting BsAb, CAR-T cells in peripheral blood increased between cycle 1 day 1 and cycle 1 day 8 in 5/7 patients with available data; median fold change in CAR-T expansion was 0.25 (25% increase) copies/ug gDNA (range -0.42-12.62). Two patients with responses to mosunetuzumab had undetectable CAR-T at baseline and developed detectable CAR-T by cycle 1 day 8. All patients who continued to receive bispecific antibodies (5/7), had detectable CAR transgene at 12 weeks to 3 months. Two patients underwent biopsy at PD; both tumors expressed CD19 and CD20 and had minimal to no infiltration by T cells. Additional samples are undergoing evaluation and will be presented at the meeting. Conclusions: Early administration of CD20 x CD3 bispecific antibodies after CAR-T appears safe and may enhance CAR-T expansion. The sequential combination of CD19 and CD20 targeted therapies may improve clinical responses in certain patients with r/r LBCL.

  PublisherOA PDFDOI

Recent grants

• NIH Grant K99CA212302

  NIH · $348k · 2019

• Resistance To Targeted Immunotherapies:CART19 as a Paradigm

  NIH · $608k · 2018–2021

Frequent coauthors

• Stephen J. Schuster

  University of Pennsylvania

  188 shared

• Elise A. Chong

  Hospital of the University of Pennsylvania

  134 shared

• Jakub Svoboda

  University of Pennsylvania

  133 shared

• Carl H. June

  Parker Institute for Cancer Immunotherapy

  130 shared

• Stefan K. Barta

  127 shared

• Sunita D. Nasta

  University of Pennsylvania

  117 shared

• Saar Gill

  University of Pennsylvania

  109 shared

• Daniel J. Landsburg

  109 shared

Labs

• Ruella LabPI

Awards & honors

• Inaugural SITC EMD Serono Cancer Immunotherapy Clinical Fell…
• AACR-Bristol Myers Squibb Oncology Fellowship in Clinical Ca…
• ASH Scholar Award (2016)
• NIH K99/R00 Pathway to Independence Award (2017)
• ISNAFF 'Paola Campese' Award for Leukemia Research (2017)