B-cell maturation antigen (BCMA)-targeted CAR T-cell therapy has shown promise in treating multiple myeloma (MM); however, relapse remains a significant challenge due to the immunosuppressive tumor microenvironment (TME) driven by Transforming Growth Factor-β (TGF-β) present in the bone marrow. To overcome this, we engineered BCMA CAR T cells armored to resist TGF-β suppression by co-expressing a dominant-negative TGF-β receptor II (DNTGF-βRII). Armored BCMA CAR T cells were manufactured with IL-7/IL-15 on the clinically compatible CliniMACS Prodigy® (Miltenyi Biotec) platform, yielding consistent transduction efficiency, expansion, and products that met all release criteria for patient dosing and infusion. The armored BCMA CAR T cells showed reduced pSmad2/3 signaling and T cell exhaustion upon TGF-β1 exposure, indicating successful resistance to TGF-β-mediated suppression, and the cells demonstrated strong cytotoxicity against primary MM tumor cells with prior BCMA exposure. In tumor models, armored BCMA CAR T cells successfully decreased or eliminated tumor burden, increased survival, and persisted in vivo. Additionally, armored BCMA CAR T-treated NSG mice showed no tumor presence after rechallenging with MM tumor cells. In summary, clinically manufactured armored BCMA CAR T cells effectively resist TGF-β-mediated immunosuppression, leading to potent anti-tumor activity in pre-clinical MM tumor models and providing supportive evidence of potential therapeutic benefit.

Abstract Introduction CAR-T therapy targeting B-cell antigens is effective for relapsed hematologic malignancies, but antigen escape remains a challenge. We previously showed dual CD19/CD20-4-1BB CAR-T therapy was highly effective (Nat Med, 2020). We further developed trispecific CAR-T cells (CD19/CD20/CD22) incorporating ICOS signaling (Sci Transl Med, 2021). Despite promising preclinical activity, trispecific-ICOS CAR-T cells failed to expand and showed no efficacy in a Phase I trial (NCT05094206; ASH 2024). Here, we investigated mechanisms underlying this failed response. Methods Based on our IRB approved protocol and using patient-derived PBMCs and leftover samples from treated patients, we generated tested bispecific-4-1BB, bispecific-ICOS and trispecific-ICOS CAR-T cells. Results NSG mice receiving bispecific- or trispecific-ICOS CAR-T cells had worse survival than those with 4-1BB CAR-T cells. In vivo, ICOS CAR-T cells failed to expand and showed an exhaustion phenotype. When co-culture with Raji cells, ICOS CAR-T cells significantly enhanced surface-FMC63 loss compared to 4-1BB counterparts. Surface-FMC63 expression was mostly recovered at 40 hours on 4-1BB CAR-T cells, but not on ICOS CAR-T cells. Since trogocytosis can mediate CAR internalization and antigen loss on tumor, we examined CD19 transfer. ICOS CAR-T cells displayed higher and more sustained CD19 uptake and fratricide reflected by apoptosis than 4-1BB cells. Consistently, when co-culture with patient autologous B cells, ICOS CAR-T cells again showed greater CAR loss and apoptosis. Extended co-culture mimicking chronic antigen stimulation revealed expansion of 4-1BB but not ICOS CAR-T cells, the latter showing reduced proliferation (Ki67) and elevated exhaustion markers. Conclusion In conclusion, the ICOS domain promotes trogocytosis and fratricide, explaining the poor persistence and function of ICOS CAR-T cells observed in our trial. Thus, our findings raise significant caution for using CAR constructs that rely on ICOS for CAR activation. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

We conducted a phase 1-2 study in which patients undergoing allogeneic hematopoietic stem cell transplantation received tildrakizumab in addition to standard immune suppression with tacrolimus and methotrexate for graft versus host disease (GVHD) prophylaxis. Fifty patients were enrolled between March 2020 and June 2023 with a median age of 56 (range 19-64). All patients received myeloablative busulfan-based conditioning and were transplanted with HLA-matched related or unrelated peripheral blood stem cell grafts. Patients were treated with tildrakizumab on an extended subcutaneous administration schedule for five doses which was well tolerated. The cumulative incidences of grades II-IV and III-IV acute graft versus host disease were 14% (95% CI 7-28) and 4% (95% CI 1-16) at day 100, respectively. The incidence of chronic GVHD requiring systemic immune suppression was 52.7% (95% CI 40.4-68.9) at twelve months. The one-year probabilities of overall, disease-free, and GVHD-free relapse-free survival were 80% (95% CI 70-92), 78% (95% CI 67-90), and 19.3% (90% CI 11.8-31.4), respectively. Pharmacokinetic analysis revealed that the half-life of tildrakizumab approximated 28 days without formation of detectable anti-tildrakizumab neutralizing antibodies. Comparative examination of fecal microbial composition in tildrakizumab and a similarly transplanted cohort treated with tocilizumab prophylaxis demonstrated that both cytokine blockade strategies had a low frequency of enterococcal dominance. We conclude that tildrakizumab resulted in a low incidence of acute GVHD and attenuation of microbiome dominance with potentially pathogenic organisms but did not mitigate the emergence of chronic GVHD as administered on this dosing schedule. NCT 04112810

Introduction: Relapsed, refractory (R/R) primary (PCSNL) and secondary central nervous system lymphoma (SCNSL) are difficult to treat diseases with poor clinical outcomes. We recently reported a high level of expansion of dual targeted lentiviral transduced anti-CD20/anti-CD19 (LV20.19) CAR T-cells into the CNS in a cohort of patients (pts) with R/R MCL (Shah et al. JCO 2025). Accordingly, we amended an ongoing single center multi-cohort clinical trial of LV20.19 CAR T cell therapy to include an arm for high-dose (HD) methotrexate R/R PCNSL or SCNSL. Methods: We conducted a Phase 1/2 single center, multi-cohort prospective trial (NCT04186520) evaluating LV20.19 CAR T-cells at a fixed dose of 2.5x10e6 cells/kg for pts with R/R B-cell non-Hodgkin Lymphoma. CAR T-cells were all manufactured on site utilizing the CliniMACS Prodigy device for a flexible 8 or 12-day manufacturing (MF) process with the goal of fresh CAR T cell infusion. Lymphodepletion was standard fludarabine/cyclophosphamide conditioning. Cohort E was added to the multi-arm trial to include pts with R/R primary or secondary CNS lymphoma involvement who had seen prior high-dose methotrexate. International Primary CNS Lymphoma Collaborative Group (IPCG) criteria from Abrey et al. JCO 2005 was used for evaluation of response. Results: To date, six pts have enrolled including four pts with PCNSL and two with SCNSL (DLBCL and MCL). All pts had parenchymal disease with enhancing lesions by MRI and two pts had concurrent leptomeningeal disease before LV20.19 CAR T cell therapy. The median age was 63 (range 42-80) and five of six patients were male. All pts received prior HD methotrexate and over 50% of pts received at least four lines of prior therapy. Three pts had prior thiotepa conditioned autologous transplants. Two pts had prior whole brain radiation. All LV20.19 CAR-T products were manufactured in 8 days, and five pts received a fresh product. One pt had his CAR T cells cryopreserved and received the product later, due to rapid CNS decline after apheresis requiring intubation and whole brain radiation. The Day 28 overall response rate (ORR) was 100% (CR=5 pts, PR=1 pt), and only one of the six patients relapsed with a median follow-up of eight months. Day 28 lumbar punctures were assessed for LV20.19 CAR T cells in four patients with all having detectable CAR T cells within the CSF. The toxicity profile of this product was in line with prior trials; 83% (n=5) experienced Grade 1-2 CRS, and no patient experienced ICANS. All pts remain alive to date. Conclusions: In this early analysis of Cohort E consisting of pts with R/R PCNSL and SCNSL, LV20.19 CAR T cell therapy demonstrates promising clinical activity with high response rates, manageable toxicity, and detectable CNS penetrance of the CAR T cells. Importantly, no patient developed ICANS and CRS was low-grade and manageable. These findings highlight the feasibility, safety, and encouraging efficacy of LV20.19 CAR T cell therapy in this high-risk patient population with historically limited treatment options and poor prognosis. This data supports further investigation of dual-targeted LV20.19 CAR T therapy in CNS lymphoma.

Introduction: Dual targeted lentiviral anti-CD20/anti-CD19 (LV20.19) CAR T cell therapy has demonstrated high overall response rates and durable efficacy in patients (pts) with relapsed, refractory B-cell NHL (Shah NN. Nature Med 2020 and Shah NN JCO 2025). As part of a multi-cohort optimization study, we evaluated both length of manufacturing and impact of cryopreservation on clinical outcomes. We report final results of pts with DLBCL/FL treated on varying arms (8 vs 12 day and fresh vs cryopreserved/thawed product). Methods: We conducted a Phase 1/2 multi-cohort prospective trial (NCT04186520) evaluating LV20.19 CAR T-cells at a fixed dose of 2.5x10e6 cells/kg for pts with R/R B-cell non-Hodgkin Lymphoma. CAR T-cells were manufactured on site utilizing the CliniMACS Prodigy device with an 8–12-day manufacturing (MF) process depending on assigned cohort. This analysis is limited to CAR naïve pts with DLBCL and FL administered either fresh CAR T-cells on the day of harvest versus a CAR T-cell product after cryopreservation/thawing. To evaluate the impact of cryopreservation on the final product, we performed seahorse metabolic profiling on 24 patients comparing oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and ATP production across apheresed T cells, fresh CAR-T cells, and post-thaw CAR-T cells. Single Cell RNA sequencing was done on 6 patients to match fresh and cryopreserved CAR-T products to assess transcriptional changes. Results: In total, 42 pts with DLBCL or FL received LV20.19 CAR T-cells. DLBCL (including transformed DLBCL and Richters Transformation) was the diagnosis in 84% of pts. All pts achieved the target cell dose. Pts were assigned as per their manufacturing arm. There were 10 pts in the fresh 8-day arm, 9 pts in the fresh 12-day arm, and 23 pts in the frozen 8-day arm. There was no difference in mean age, sex, and lines of prior therapy between the fresh and cryopreserved/thawed cohorts. The ORR/CR for 8-day fresh vs 12-day fresh was 90%/70% vs 78%/22%, p=0.58 (ORR), and for 8-day fresh vs 8-day frozen was 90%/70% vs 74%/57%, p=0.40 (ORR). The 1-year PFS was 40% for 8-day fresh vs 22% for 12-day fresh vs 55% for 8-day frozen cohorts (p=0.08). Similarly, 1-year OS was 70% for 8-day fresh vs 56% for 12-day fresh vs 76% for 8-day frozen (p=0.18). Among treated pts any grade CRS occurred in 79%, with 1 pt experiencing Grade 3 CRS in the 8-day fresh cohort. There was a trend towards more frequent CRS in 8-day fresh products vs 8-day frozen (100% vs 74%, p=0.09). In total, 24% of pts experience any grade ICANS but only 2 pts (5%) had grade 3 ICANS with no difference among the cohorts. Correlative analyses performed on fresh and post-thaw CAR-T cells demonstrated significantly reduced OCR, ECAR, mitochondrial and glycolytic ATP production, maximal respiratory capacity, and spare respiratory capacity in the cryopreserved cohort (p<0.01). Transcriptomic analysis revealed downregulation of key cytotoxic and activation markers (IFNG, GZMB, GZMA, PRF1, NKG7), along with upregulation of genes associated with senescence and apoptosis in cryopreserved samples (p<0.05). Importantly, no major metabolic differences were observed between apheresed T cells and fresh CAR-T cells, indicating that cryopreservation—rather than manufacturing—drives the observed functional decline observed immediately post-thaw. Differentiation immunophenotyping of final CAR-T cell products showed that both CD4 and CD8 T CAR-T cell subsets were more terminally differentiated in 12-day products versus 8-day products (p<0.005). Conclusions: Through this multi-cohort optimization of LV20.19 CAR T cell study we did not see clinical differences among fresh versus cryopreserved treated pts; however, there were trends towards worse outcomes in pts with 12-day MF vs 8-day MF. ORR/CR were highest for 8-day fresh pts at 90%/70% although this did come with slightly higher rates of low-grade CRS. Correlative studies demonstrated diminished metabolism in LV20.19 CAR T cells post-cryopreservation with upregulation of genes associated with senescence and apoptosis. Immunophenotyping revealed improved differentiation profile of shorter manufactured cells. Taken together, these data suggest a short 8-day MF process with a fresh infusion is optimal for LV20.19 CAR T cell therapy, and this platform was subsequently utilized for all future arms on this clinical trial.

BACKGROUND:High-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival. Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in HR-NBL patients; however, graft-versus-host-disease (GVHD) and disease progression have limited clinical application. Ex-vivo stimulated allogeneic natural killer (NK) cells represent a potential approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL. METHODS:Ex-vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble IL-15/IL-15Rα alone or with irradiated CD137L/CD54+ AgN2a-4P (15-4P) at a 1:1 ratio for 10-12 days. Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-TIM-3. Lethally irradiated B6AJF1 Mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15-4P B6 NK cells on days 14, 21, and 28. In select experiments, T cell and NK cells were selectively depleted to establish their contribution to the GVT effect. All groups were analyzed for tumor growth, GVHD and overall survival. RESULTS:Co-culturing NK cells with 15-4P results in 78-fold expansion with increased expression of Ki-67 and NKG2D, NKp46, TRAIL and TIM-3. 15-4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared to IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared to NXS2. The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15-4P NK cell degranulation, Eomes, TRAIL and FasL expression observed. Analysis of RNA from 15-4P NK cells exposed to TIM-3 blockade showed gene expression of chemokines, NKG2D/DAP12 signaling, non-canonical NF-κb pathway and TRAIL signaling. Blockade of NKG2D, TRAIL or FasL on 15-4P NK cells abrogated cytotoxicity. In vivo, the combination of 15-4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared to NK cells or anti-TIM-3 alone, without inducing GVHD. Depletion of NK cells, but not T cells, abrogated the GVT effect. CONCLUSION:Allo-HCT can be a platform for treating NBL using combination ex-vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD.

Background CAR-T cell therapy targeting B-cell antigens has become an established strategy for relapsed hematological malignancies. However, development of even more effective CAR-T therapy to overcome antigen-escape relapse is warranted. We previously demonstrated that dual targeted CD19/CD20-4-1BB CAR-T therapy was highly effective (Nat Med, 2020). To further improve outcome, we generated trispecific duo CAR-T cells (CD19/CD20/CD22) with tandem anti-CD20/CD19 CD3z/ICOS intracellular signaling domains (ICD) and a separate anti-CD22 scFv with CD3z alone. This construct exhibited efficient T-cell signal transduction and tumor control in vitro and in NSG mice (Sci Trans Med, 2021). Surprisingly, trispecific-ICOS CAR-T cells failed to expand in patients and thus lacked therapeutic efficacy in a Phase I (NCT05094206) clinical trial (ASH 2024). In this study, we examined the potential mechanisms underlying the failed response of trispecific-ICOS CAR-T cells. Methods Based on our IRB approved protocol and using patient-derived PBMCs and leftover samples from treated patients, we generated tested bispecific-4-1BB, bispecific-ICOS and trispecific-ICOS CAR-T cells, and tested their activation and function. In vivo xeno-graft mouse models were used to better understand CAR kinetics, response, and outcomes. Results We first examined the ability of CAR-T cells to control Raji cell growth in an in vivo mouse model using a lower, more clinically relevant, dose than what had been used in prior studies. Recipients of bispecific- or trispecific-ICOS CAR-T cells showed worse survival compared to those of bispecific 4-1BB CAR-T cells. Similarly, ICOS CAR-T cells failed to expand and exhibited an exhaustion phenotype in Raji-bearing mice. Since ICOS and 4-1BB CAR-T cells expanded similarly during manufacture with IL-7/IL-15 cytokines, we hypothesized that ICOS CAR-T cells may be defective in their ability to response to antigen. To test this, we co-cultured CAR-T cells with Raji cells for 1, 5, 15 and 40 hours, and found that these CAR-T cells quickly down-regulated CAR expression as reflected by loss of surface FMC63 staining. Given that total FMC63 levels were not reduced in any type of CAR-T cells through surface and intracellular staining, these results indicate that CAR was internalized. Strikingly, ICOS CAR-T cells significantly enhanced surface-FMC63 loss compared to 4-1BB counterparts after culture for 1, 5 and 15 hours. Surface-FMC63 expression was mostly recovered at 40 hours on 4-1BB CAR-T cells, but not on ICOS CAR-T cells. Trogocytosis, a process whereby some proteins from one cell are transferred to another cell, has been reported to occur between CAR-T cells and their targeted tumor cells contributing to antigen loss on tumor cells and fratricide CAR T-cell killing. We thus examined CD19 expression on CAR-T cells after Raji stimulation and observed transient expression of CD19 on 4-1BB CAR-T cells, suggesting that low level trogocytosis was occurring. Surprisingly, ICOS-CAR T cells demonstrated higher degree and more sustained surface CD19 expression as well as increased apoptosis, suggesting augmented fratricidal T-cell killing. To exclude the impact of allogeneic responses to Raji cells, we purified B cells from PBMCs derived from the same patient as the CAR-T cells. Consistent with the Raji cells, ICOS-CAR-T cells showed significantly decreased surface-FMC63 and increased apoptosis compared to 4-1BB CAR-T cells after co-culture with autologous B cells. Taken together, these data indicate that ICOS ICD enhanced trogocytosis and fratricide of CAR-T cells. We further co-cultured CAR-T and Raji cells for 2 weeks, to mimic the in vivo condition where CAR-T cells are continuously stimulated by lymphoma cells in patients. While 4-1BB CAR-T cells expanded during the culture period, ICOS CAR-T cells declined in numbers. ICOS CAR-T cells exhibited decreased proliferation (Ki67) and increased markers of exhaustion (Tim3, PD-1 and Lag3). These results indicate trogocytosis as the key mechanism underlying failed expansion and lack of response of ICOS CAR-T cells in our clinical trial. Conclusions In conclusion, we found that ICOS ICD signal drives strong trogocytosis and promotes surface expression of tumor antigen that prevents CAR-T cells from expanding properly. Thus, our findings raise significant caution for using CAR constructs that rely on ICOS for CAR activation.

Mitochondria, as regulators of cellular energy production and metabolism, play a crucial role in tumor growth and survival. Tumors are reprogrammed to accommodate rapid proliferation through the Warburg effect. This reprogramming leads to the accumulation of metabolites such as lactate and ketone bodies, thereby lowering the pH of the tumor microenvironment, inhibiting the activity of effector T cells and NK cells, while promoting the infiltration of regulatory T cells and MDSCs, forming an immunosuppressive microenvironment. ROS produced by mitochondria can affect immune cell function by modulating their signaling pathways. Mitochondria also release DAMPs, which activate the antigen-presenting capacity of dendritic cells and initiate anti-tumor immune responses. Currently, various methods have been employed, such as DLCs modifications and mitochondrial targeted delivery, which enable drugs to penetrate the lipid bilayer and enter the mitochondria, thereby helping to reduce immunosuppression in the tumor microenvironment. In this review, we will discuss the impact of mitochondria on tumor immunity, strategies to target tumor cell mitochondria, and progress on the discovery of mitochondria-targeted drugs to enhance tumor immunity, providing potential directions for developing new cancer therapeutic strategies.

PURPOSE:Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy characterized by t(11;14) and bright CD20 expression. To improve outcomes from single targeted CD19 chimeric antigen receptor (CAR) T cells, we used dual targeted lentiviral anti-CD20/anti-CD19 (LV20.19) CAR T cells as part of a phase I/II clinical trial in relapsed, refractory (R/R) MCL (ClinicalTrials.gov identifier: NCT04186520). METHODS:Patients with MCL who had failed two lines of therapy or relapsed post-transplant were eligible. LV20.19 CAR T cells were manufactured on-site via CliniMACS Prodigy using an adaptive 8- to 12-day process to optimize the final CAR product for increased numbers of naïve and stem-cell memory (SCM) like T cells. RESULTS:Seventeen patients with R/R MCL received a single dose of LV20.19 CAR T cells at 2.5 × 106 cells/kg (phase I = three patients; phase II = 14 patients). The best overall response rate (ORR) was 100% (complete response [CR] = 88%; partial response = 12%) and the phase II efficacy threshold for day-90 CR rate was exceeded. Two patients have relapsed as of the data cutoff and neither the median progression-free survival nor overall survival has been reached with a median follow-up of 15.8 months. Ninety-four percent (n = 16) experienced cytokine release syndrome, all grade 1-2. Eighteen percent (n = 3) had immune effector cell-associated neurotoxicity syndrome in the first 28-days, two with reversible grade 3 toxicity. Three patients had nonrelapse mortality events; all occurred in the setting of ongoing B-cell aplasia. The final LV20.19 CAR products were enriched for higher percentages of T-SCM/T-naïve cells and most patients received CAR T cells within 8 days of apheresis. CONCLUSION:In conclusion, we demonstrate that on-site adaptive manufactured LV20.19 CAR T cells are feasible, safe, and efficacious for R/R MCL with best ORR of 100%, a favorable safety profile, and few relapses to date.

Study representation of underserved communities

BackgroundAlloHSCT serves as a long-term curative intervention for CAYA with varied disorders but poses risks like refractory ADV infections due to impaired T-cell immunity (George/Cairo, BJH, 2012). Here, we report data from the Viral Cytotoxic T-Lymphocyte Consortium (VIRCTLC), showing the safety and efficacy of employing familial ADV-specific CTLs in AlloHSCT recipients.ObjectiveAssess safety and effectiveness of familial ADV-specific CTLs for refractory ADV post-AlloHSCT.Design/MethodsPatients post-AlloHSCT showing refractory ADV were eligible. Refractory ADV infection was defined by increasing serum RT-PCR DNA (by 1 log) after 7 days or persistent quantitative RT-PCR DNA copies after 14 days of appropriate anti-viral therapy, and/or known resistance/intolerance to anti-viral agents. Related donors were matched at ≥3 HLA (A, B, or DRB1) loci and had an adequate T-cell IFN-γ response to ADV specific MACS PepTivators®. Direct selection was used to expedite cell product generation, in a point-of-care setting. Donor peripheral blood mononuclear cells were collected using non-mobilized apheresis. ADVCTLs were isolated using the CliniMACS ® Prodigy following stimulation with specific MACS® GMP PepTivator® AdV5 Hexon, provided by Miltenyi Biotec®. ADV-specific CTLs were enriched using a Cytokine Capture System (CCS). Target cell dose was 0.5 × 104 CD3+ cells/kg (recipient weight) for haploidentical related donors and 2.5 × 104 CD3+ cells/kg for matched related donors. Repeated doses were permitted every 2 weeks in the absence of a complete response (CR) and adverse events. The following were used to define response after initial 5 doses: CR - undetected ADV PCR, partial response (PR) - at least one log decrease from baseline, progressive disease (PD) – at least one log increase from baseline, and patients with stable disease.ResultsOf 15 enrolled patients (10F, 5M; ages 1-19), all post-AlloHSCT, 8 vsCTLs were derived from the original familial HSCT donors, and 7 from third-party donors (5 maternal, 2 paternal). The mean number of ADV CTL infusions was 3.7 (range: 1-16). Thirteen patients achieved CR as defined and 2 achieved PR. The overall response (OR) was 100% and the CR was 88%. The average time to OR was 33 days (range 6-112 days). Day 100 and 365 overall survival post-HSCT was 86.1% (CI95: 49.4-95.7) and 70.5% (CI95: 38.9-87.8), respectively (Figure 1). Among CR patients, Day 100 and 365 survival was 83.9% (CI95: 55.0-94.3) and 74.6% (CI95: 39.8-91.1), respectively. ADV-related mortality at both time points was 0% (Figure 2). One patient developed acute grade 2 skin GVHD possibly related to infusion, which resolved.ConclusionPreliminary data affirm that donor ADV-specific CTLs are safe and effective in treating refractory ADV cases post-AlloHSCT. Manufacturing is rapid and reliable. Supported by FDA RO1006301A1. Enrollment is ongoing.

IntroductionDespite advances in targeted therapies, outcomes for both relapsed/refractory (R/R) CLL and RT remain poor. As part of a multi-cohort Phase 1/2 trial of lentiviral bispecific anti-CD20/anti-CD19 (LV20.19) CAR T cells for R/R B cell malignancies (NCT04186520), patients (pts) with RT were allowed to enroll on a DLBCL cohort and pts with CLL were enrolled on a dedicated Phase 1 arm. We report below initial safety and outcomes with LV20.19 CAR for CLL and RT pts.MethodsWe conducted a Phase 1/2 single center, prospective trial of LV20.19 CAR T cells at a fixed dose of 2.5 × 10e6 cells/kg manufactured utilizing an adaptive 8/12 day process with CliniMACS Prodigy. Eligible RT pts must have failed two lines of RT directed treatment. CLL pts required two prior lines of therapy, one mandated to be BTK or BCL2 inhibitor. Lugano criteria was utilized for RT response while Hallek iwCLL criteria was used for nodal CLL response.Results14 pts with R/R RT and CLL received LV20.19 CAR T cells (RT=4 and CLL=10). All pts attained target cell dose. Median age was 65 years (55-75) and 12/14 pts were male (86%). Median lines of prior therapy was 3 (range 2-10). 12 pts (86%) had disease progression on covalent BTKi and venetoclax. Tp53 aberrations were present in 6 pts (43%) and 8 pts (57%) had complex cytogenetics.In terms of safety, 100% (n=14) developed CRS and 93% (n=13) required tocilizumab. There were 2 pts with grade 3 CRS, both with CLL. ICANS occurred in 3 pts (21%); 1 CLL patient (pt) had grade 3 ICANS and another CLL pt had grade 4 ICANS, the latter was found to have CLL involvement in the CSF. ICE-HS (immune effector cell hemophagocytic lymphohistiocytosis like syndrome) occurred at high rates developing in 9 pts (64%). Two CLL pts had grade 3 and grade 4 IEC-HS, respectively. Both met criteria for a dose limiting toxicity (DLT); one died of fungemia related to immunosuppression before disease assessment. The median ferritin in IEC-HS pts was 22454 ng/mL (range 8091-183578). Six (66%) IEC-HS pts required anakinra for management. Among CLL pts, there was no difference in CAR expansion (Figure 1) or polyfunctional strength index of the final product (Figure 2) between pts who did and did not develop IEC-HS. The 1-year non-relapse mortality was 7%.The day 28 ORR for evaluable pts (n=13) was 92% (CR=46%, PR=46%). In responding pts (n=12), day 28 bone marrow was negative for disease. To date, among these 12 pts, only 1 RT pt has relapsed with a median follow-up time of 11 months. The median duration of response was not reached (Figure 3) and the median overall survival was 15 months (Figure 4).ConclusionsWhile LV20.19 CAR T cells were efficacious in CLL and RT it was limited by high rates of IEC-HS especially among CLL pts, a phenomena not commonly seen with other histologies. With two DLTs in the CLL cohort, the dose for future CLL pts was reduced to 1 × 10e6 cells/kg. Additional studies are needed to understand how CLL biology drives CAR IEC-HS.

Abstract Purpose: Vaccination with dendritic cell (DC)/multiple myeloma (MM) fusions has been shown to induce the expansion of circulating MM-reactive lymphocytes and consolidation of clinical response following autologous hematopoietic cell transplant (autoHCT). Patients and Methods: In this randomized phase II trial (NCT02728102), we assessed the effect of DC/MM fusion vaccination, GM-CSF, and lenalidomide maintenance as compared to control arms of GM-CSF and lenalidomide or lenalidomide maintenance alone on clinical response rates and induction of MM-specific immunity at 1-year post-transplant. Results: The study enrolled 203 patients, with 140 randomized post-transplantation. Vaccine production was successful in 63/68 patients. At 1 year, rates of CR were 52.9% (vaccine) and 50% (control) (p=0.37, 80% CI 44.5%, 61.3% and 41.6%, 58.4%, respectively), and rates of VGPR or better were 85.3% (vaccine) and 77.8% (control) (p=0.2). Conversion to CR at 1 year was 34.8% (vaccine) and 27.3% (control) (p=0.4). Vaccination induced a statistically significant expansion of MM-reactive T cells at 1 year as compared to prior to vaccination (p=0.024) and in contrast to the non-vaccine arm (p=0.026). Single-cell transcriptomics revealed clonotypic expansion of activated CD8 cells and shared dominant clonotypes between patients at 1-year post-transplant. Conclusions: DC/MM fusion vaccination with lenalidomide did not result in a statistically significant increase in CR rates at 1-year post-transplant but was associated with a significant increase in circulating MM-reactive lymphocytes indicative of tumor-specific immunity. Site-specific production of a personalized cell therapy with centralized product characterization was effectively accomplished in the context of a multicenter cooperative group study.

Introduction: CD19 based CAR T cell therapy has become a standard of care treatment for patients with relapsed B-cell malignancies. Recently there has been increased focus on the development of secondary malignancies post CAR T cell therapy. As part of completed and ongoing clinical trials with bispecific LV20.19 CAR T cells at the Medical College of Wisconsin, all patients underwent a baseline bone marrow biopsy and at minimum repeat biopsy at day 28. Those with marrow involvement had repeat bone marrow biopsies at pre-specified times points until 2-years post-CAR T. We now evaluate these patients for development of new cytogenetic abnormalities post CAR T cell therapy as part of long-term follow-up of these trial patients. Methods: We performed a retrospective review of patients who received a next generation bispecific (LV20.19) CAR T-cell product on clinical trial (NCT03019055, NCT04186520). All patients received fludarabine and cyclophosphamide combinations as part of lymphodepletion. A baseline marrow aspirate and biopsy prior to CAR T and on day 28 after CAR T therapy was obtained on all patients. We evaluated the presence of cytogenetic changes found on all bone marrow samples collected following CAR T therapy. Adults with R/R diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL) were included. Descriptive and survival analyses using the Kaplan-Meier methodology were performed. Median overall survival (OS) was estimated from the date of CAR T treatment to death or last follow-up. Results: From November 1, 2017 to July 1, 2024 a total of 96 patients who received LV20.19 CAR T therapy on clinical trials were evaluated for presence of cytogenetic abnormalities post-CAR. We identified 21 patients (22%) with new cytogenetic abnormalities after LV20.19 CAR T cell therapy. Among this subset, the median age was 64 years (46-75) and 86% (n=18) were male sex. The most common B-cell malignancy was DLBCL in 43% of patients (n=9) followed by CLL in 28% (n=6). 8 patients (38%) had a prior autologous stem cell transplant. The most common cytogenetic findings identified included loss of Y in 6 patients and abnormalities within chromosome 7 (including translocation, loss of 7, and del 7q) in 8 patients. Other abnormalities identified included del(5q) (n=1), del(20q) (n=1), t(1;12) with del(2) (n=1), t(5;19) (n=1), t(3;6) (n=1), +15 +Y (n=1), del(1) (n=1). The cytogenetic abnormalities disappeared on subsequent marrows in patients with 7q (n=1), t(7;14) n=1, t(1;12) and del 2 (n=1), and +15 (n=1). The median time from LV20.19 CAR T cell therapy to discovery of the abnormality was 8 months (1 month-24 months). Despite the presence of cytogenetic abnormalities only 5 of the 21 patients developed morphologic evidence of myelodysplastic syndrome. 4 of the 5 patients passed due to either MDS related or allogeneic transplant related complications. The median OS for the 21 patients with cytogenetic abnormalities was 60 months. Conclusions Cytogenetic abnormalities were common in the bone marrow in patients receiving LV20.19 CAR T cell therapy as part of a clinical trial. However, most findings did not correlate with development of a new hematological malignancy, and others were transient in nature with no clinical consequence. These data demonstrate the importance of long-term follow-up of CAR T cell therapy patients given evolving cytogenetic abnormalities post-treatment.

Background & AimCAR-T cell therapy continues to be a powerful approach for treating relapsed, refractory hematological malignancies. Cryopreservation of CAR-T cell products prior to administration is standard practice in the delivery process between third-party manufacturing sites. At the Medical College of Wisconsin, utilizing a point of care CAR-T manufacturing system (CliniMACS Prodigy ®), the goal has been to administer fresh, non-cryopreserved bispecific, lentiviral anti-CD20, anti-CD19 (LV20.19) CAR-T to all patients when clinically feasible. To determine if cryopreservation affects the final CAR product we initiated a new arm on an ongoing clinical trial (NCT04186520) that mandates cryopreservation prior to infusion.Methods, Results & ConclusionWe initiated a 24-patient arm within an ongoing trial utilizing LV20.19 CAR-T cells for patients with relapsed, refractory B-cell DLBCL, FL, and MZL. The manufacturing of this arm mirrored our fresh cohorts but mandated cryopreservation of the final product before infusion. To assess whether cryopreservation affected the activation or bioenergetic capacity of administered CAR-T cells, we examined energy metabolism and activation status of T cells prior to CAR manufacturing, of CAR-T cells immediately before cryopreservation (“fresh”), and of CAR-T cells after thawing on the day of infusion (“post-thaw”) from the same patient, using Seahorse Pro analyzer to measure oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and ATP production.Analysis of 15 intra-patient comparisons indicated statistically significant decreases in both the OCR and ECAR in the post-thaw CAR-T cells. Moreover, maximal respiratory capacity and spare respiratory capacity (SRC) were significantly decreased in the post-thaw CAR-T cells. Total glycolytic and mitochondrial ATP production decreased in post-thaw CAR-T cells compared to fresh CAR-T cells.Together these data suggest that cryopreservation impacts CAR-T-cell energy metabolism and activation. The decrease in total ATP production in post-thaw samples suggests impaired CAR-T cell functionality. However, to date, the clinical significance of these findings remains unknown. We continue to enroll patients to this cryopreservation cohort with plans to perform comparative analyses with patients infused with fresh product. Specifically, we will compare in vivo expansion, severity, and time to CRS and ICANS, along with overall response and duration of response.

Supplemental Figure 1: (A) Progression Free Survival Kaplan Meier Curve. (B) Overall Survival Kaplan Meier Curve.

7043 Background: Combination trials of BTK inhibitors (BTKi) with CAR-T have suggested potential synergistic benefit by improving CAR-T immunophenotype and clinical outcomes but are limited by increased toxicity. We report immunophenotype, cytokine profile, and patient outcomes with the new non-covalent BTKi, pirtobrutinib (pirto) when given prior to LV20.19 CAR-T and compare the final CAR product to pts who did not receive pirtobrutinib prior to CAR. Methods: Pts received LV20.19 CAR-T as part of a phase 1/2 trial (NCT04186520). Only pts who received pirto £4 weeks prior to apheresis were included. To determine the impact of pirto on LV20.19 CAR-T, we calculated both polyfunctionality (PFA) and polyfunctional strength index (PSI) for each product using the Isoplexis. Descriptive statistics, t-tests and Kaplan-Meier method were used as appropriate. Results: 11 pts received pirto prior to LV20.19 CAR-T (Table). Median age was 65 (50-80) yrs and median prior lines of therapy were 4 (2-8). There were 4 MCL pts, 5 Richter’s (RT)/CLL, 1 MZL and 1 DLBCL. Median duration of pirto was 4 (1-20) mo. Pts were on pirto for a median of 12 (1-22) days prior to apheresis. All except 1 pts received target CAR-T dose. The day 28 ORR was 82% (CR=7, PR=2). After a median follow up of 13 mo, 3 pts died (PD, Covid, Guillain Barre). The median PFS and OS were both 30.8 mo while the 1-year PFS and OS rates were 9 and 15 mo respectively. 9 pts had CRS and 2 had ICANS, all grades 1-2, while 4 had IEC-HS. One pt had afib recurrence and 1 pt had CMV viremia within 30 days of CAR-T. Among the pts with immunophenotypic data (n=10), there were no differences in naïve or more differentiated T-cell percentages in apheresis and final CAR-T products when compared to pts who did not receive pirto before LV20.19 CAR-T (n=57). Although not statistically significant, both PFA (CD4=61.8 vs 59, CD8= 49.8 vs 46.9) and PSI (CD4= 1752 vs 1608, CD8=1299 vs 1079) were higher with pirto pre-treated pts, suggesting a potential trend towards improved CAR-T functionality. Conclusions: These data represent the largest experience of pirto prior to CAR-T apheresis and demonstrate that pirto can be safely used as a bridge to LV20.19 CAR-T without negatively impacting their immunophenotype and potentially improving functionality. These data support our planned phase 1 clinical trial to assess the safety of pirto as bridging and maintenance therapy with LV20.19 CAR-T (NCT05990465). [Table: see text]