B-cell Maturation Antigen Chimeric Antigen Receptor Research Articles

CD4+ CAR T-cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR T-cell therapy

AbstractMultiple myeloma is characterized by frequent clinical relapses after conventional therapy. Recently, chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, although >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune-intrinsic mechanisms may contribute to this resistance. Although there were no preexisting T-cell features associated with clinical outcomes, we found that patients with a durable response to CAR T-cell treatment had greater persistence of their CAR T cells than patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR T cells. These cells expand in vivo early after infusion but express exhaustion markers (Hepatitis A virus cellular receptor 2 [HAVCR2] and T cell immunoglobulin and mucin domain-containing-3 [TIGIT]) and remain polyclonal. Finally, we demonstrate that nonclassical monocytes are enriched in the myeloma niche and may induce CAR T-cell dysfunction through mechanisms that include transforming growth factor β. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR T-cell therapy.

Abstract 3616: In vivo expansion patterns of anti-BCMA CAR T-cell in relapsed/refractory multiple myeloma: Comparative immunomonitoring of idecabtagene vicleucel and ciltacabtagene autoleucel

Abstract Introduction: Chimeric antigen receptor (CAR) T cell (CAR-T) treatments have revolutionized the treatment of multiple myeloma (MM). Two CAR-T cell products targeting B cell maturation antigen (BCMA) have received approval for the treatment of MM: idecabtagene vicleucel (idecel) and ciltacabtagene autoleucel (ciltacel). A comparative analysis of in vivo CAR-T responses in patients receiving these novel cellular therapies has not been performed, especially not in the real-world setting. Methods: We prospectively enrolled 23 MM patients treated with idecel or ciltacel in our study GCCC2043. Blood samples were obtained at apheresis, pre-lymphodepleting (LD) chemotherapy, on days 0, +7, +14, +21, +28 and at +3 months post CAR-T. Samples were stained using BCMA or control CAR detection reagents, monoclonal antibodies against T cell surface antigens, and analyzed using flow cytometry. Results: Baseline clinical characteristics, pre-apheresis and pre-LD absolute lymphocyte counts were comparable between groups. There were no G3 cytokine release syndrome (CRS) events whereas G1/2 CRS and all-grade Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) events were not significantly different between cohorts. The global overall response rate (ORR) was 78.3% (n=18) and 60.9% (n=14) at days 30 and 90, respectively. At +1 month, 72.7% (8/11) of idecel and 83.3% (10/12) of ciltacel cohort had an objective response (p=0.625). There was no statistical ORR difference between idecel and ciltacel cohorts at 3, 6, and 9 months. Two-way ANOVA of CAR-T expansion studies revealed a trend towards an effect of treatment type (p=0.0595). Time to peak CAR-T numbers was significantly shorter for idecel (p=0.0127), however, ciltacel showed a significantly more pronounced CAR-T expansion (p=0.043) and longer persistence. Peak CAR-T expansion was associated with clinical responses across cohorts at +1 (p=0.034) and +3 months (p=0.050). Day +30 responses were indicative of Day +90 and Day +180 responses (p<0.001, p=0.011). There was no difference in CD8+ CAR T cell peak levels, however, CD4+ CAR T cell peak levels were much higher (p=0.019) for ciltacel vs. idecel. Similarly, there were significantly higher (p=0.002) CD4+/CD8+ double-positive CAR-T peak levels in ciltacel vs. idecel patients. There was no significant difference in the distribution of T cell memory subtypes at peak level. Conclusions: We demonstrate here for the first time significant differences in CAR T cell expansion and persistence patterns following infusion of either idacel or ciltacel. Studies are underway at our institution investigating anti-BCMA CAR T cell responses in more detail and larger numbers of patients. Such studies have the potential to identify predictive/prognostic biomarkers and/or lead to further optimization of myeloma-targeting CAR-T. Citation Format: Mehmet Kocoglu, Aaron P. Rapoport, Etse Gebru, Destiny Omili, Daniel Yamoah, Rediet Mulatu, Jean Yared, Nancy M. Hardy, Michael Kallen, Ashraf Z. Badros, Djordje Atanackovic. In vivo expansion patterns of anti-BCMA CAR T-cell in relapsed/refractory multiple myeloma: Comparative immunomonitoring of idecabtagene vicleucel and ciltacabtagene autoleucel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3616.

Abstract 3617: Expansion and persistence of anti-BCMA CAR T cells correlates with durability of responses in multiple myeloma patients

Abstract Background: Chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA) can induce >70% response rates in patients with relapsed/refractory multiple myeloma (RRMM). However, most patients ultimately relapse. There are two FDA-approved CAR T products: ciltacabtagene autoleucel (cilta-cel) and idecabtagene vicleucel (ide-cel), the former having much more durable responses. The mechanisms that mediate more durable responses with these CAR T cells is unknown. Previous studies have shown that specific cell states in the apheresis and infusion products are associated with long-term efficacy of anti-BCMA CAR therapy in MM and the relationship between durability of response and CAR T cell persistence remains unclear. Methods: We analyzed serial bone marrow and PBMC samples from 20 MM patients who received BCMA CAR T therapy, including cilta-cel, ide-cel, and JCARH125. Flow cytometry and single-cell RNAseq were used to evaluate the features of CAR T cells that are associated with durable clinical responses. Results: While CAR T cells were detectable but variable among patients at 1-month post infusion, the frequency of CAR T cells in bone marrow at 1-month post-infusion is positively correlated with progression free survival (PFS) in all MM patients (p=0.009). This association was also evident in patients just treated with cilta-cel (p= 0.008). By 6 months, CAR T cells could still be detected by high-throughput flowcytometry in the cilta-cel-treated patients, but CAR T cells were not detectable in most of the patients receiving the other 2 products. Cilta-cel CAR T cells predominantly possessed an effector-like surface phenotype at 1-moth post infusion in both CD8+ and CD4+ compartments in bone marrow. These CAR T cells shifted to effector memory and central memory T cell states by 6 months, respectively. For ide-cel and JCARH125, patients with more durable responses (>6mos) there was a significantly higher proportion of CAR T cells with CD8+ effector memory-like state at 1-month post infusion. Conclusions: Our data demonstrate that distinct anti-BCMA CAR T cell treatments lead to very different cellular outcomes: Cilta-cel CAR T cell treatment leads to greater expansion and persistence compared to other anti-BCMA CAR T cells. Cilta-cel CAR T cells also shift from the initial effector state to a memory phenotype. These results provide insights into features of more efficacious anti-BCMA CAR T cells that can help guide the future development of more durable treatments for MM. Citation Format: Kai Wu, Karen Law, Guy Ledergor, Chang Liu, Zenghua Fan, Vibha Gurunathan, Averey Lea, Matthew Clark, Serena Kwek, Alexander Cheung, Jeffrey Wolf, Ajai Chari, Anupuma Kumar, Justin Eyquem, Thomas Martin, Lawrence Fong. Expansion and persistence of anti-BCMA CAR T cells correlates with durability of responses in multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3617.

Antigen-induced chimeric antigen receptor multimerization amplifies on-tumor cytotoxicity

Ligand-induced receptor dimerization or oligomerization is a widespread mechanism for ensuring communication specificity, safeguarding receptor activation, and facilitating amplification of signal transduction across the cellular membrane. However, cell-surface antigen-induced multimerization (dubbed AIM herein) has not yet been consciously leveraged in chimeric antigen receptor (CAR) engineering for enriching T cell-based therapies. We co-developed ciltacabtagene autoleucel (cilta-cel), whose CAR incorporates two B-cell maturation antigen (BCMA)-targeted nanobodies in tandem, for treating multiple myeloma. Here we elucidated a structural and functional model in which BCMA-induced cilta-cel CAR multimerization amplifies myeloma-targeted T cell-mediated cytotoxicity. Crystallographic analysis of BCMA–nanobody complexes revealed atomic details of antigen–antibody hetero-multimerization whilst analytical ultracentrifugation and small-angle X-ray scattering characterized interdependent BCMA apposition and CAR juxtaposition in solution. BCMA-induced nanobody CAR multimerization enhanced cytotoxicity, alongside elevated immune synapse formation and cytotoxicity-mediating cytokine release, towards myeloma-derived cells. Our results provide a framework for contemplating the AIM approach in designing next-generation CARs.

NK92 Expressing Anti-BCMA CAR and Secreted TRAIL for the Treatment of Multiple Myeloma: Preliminary In Vitro Assessment.

Multiple myeloma (MM) has witnessed improved patient outcomes through advancements in therapeutic approaches. Notably, allogeneic stem cell transplantation, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies have contributed to enhanced quality of life. Recently, a promising avenue has emerged with chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA), expressed widely on MM cells. To mitigate risks associated with allogenic T cells, we investigated the potential of BCMA CAR expression in natural killer cells (NKs), known for potent cytotoxicity and minimal side effects. Using the NK-92 cell line, we co-expressed BCMA CAR and soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) employing the piggyBac transposon system. Engineered NK cells (CAR-NK-92-TRAIL) demonstrated robust cytotoxicity against a panel of MM cell lines and primary patient samples, outperforming unmodified NK-92 cells with a mean difference in viability of 45.1% (±26.1%, depending on the target cell line). Combination therapy was explored with the proteasome inhibitor bortezomib (BZ) and γ-secretase inhibitors (GSIs), leading to a significant synergistic effect in combination with CAR-NK-92-TRAIL cells. This synergy was evident in cytotoxicity assays where a notable decrease in MM cell viability was observed in combinatorial therapy compared to single treatment. In summary, our study demonstrates the therapeutic potential of the CAR-NK-92-TRAIL cells for the treatment of MM. The synergistic impact of combining these engineered NK cells with BZ and GSI supports further development of allogeneic CAR-based products for effective MM therapy.

Heme Attenuates T Cell Exhaustion and Drives Effector Function: Implications for Immune and Adoptive T Cell Therapy

Introduction: Though frontline agents have been effective in multiple myeloma (MM) therapy, a significant proportion of MM patients relapse and succumb to refractory disease. B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T therapy represents an exciting novel therapeutic strategy for MM but still suffers from limited in vivo T cell expansion and CAR T cytotoxicity. Altering the CAR T manufacturing process to produce CAR T cells with less exhausted phenotypes and robust killing is an active area of research. Mitochondrial function and energetics play a central role in regulating T cell fate and function, and heme (iron-protoporphyrin IX) is critical for maintaining electron transport chain activity and oxidative phosphorylation. Heme can be sourced from the extracellular milieu, diet, or intrinsic heme biosynthesis and has a multitude of cellular functions throughserving as a cofactor for enzymatic and metabolic reactions. Additionally, heme is catabolized by heme oxygenase 1, producing the antioxidants biliverdin and bilirubin critical to preserving cellular redox homeostasis. In the present study, we investigate the effects of heme on CAR T cell efficacy. Our results suggest that heme supplementation during ex vivo CAR T manufacturing or supplementation during CAR T-tumor cell engagement can lead to decreased T cell exhaustion and increased tumor killing. Methods: Human healthy donor and MM patient T cells were activated and expanded with α-CD3/CD28 and IL-2 for 7 days +/- hemin, after which immunostaining and metabolic assays were conducted. For CAR T manufacture, T cells were transduced with CAR construct starting on day 3 and were co-cultured with antigen-expressing tumor cells in vitro on day 7-8. Cytotoxicity and cytokine production were assayed after 24 hours of co-culture. Results: In a dose-dependent manner, heme supplementation significantly decreases proportions of LAG3 + and TIM3 + and decreases proportions of CD62L + and TCF1 + cells in both CD4 + and CD8 + subsets isolated from healthy donor and MM patient peripheral blood. Supportive of these observations, inhibition of de novo heme biosynthesis leads to the opposite effect with an increase in proportions of PD1 +, LAG3 +, TIM3 +, and CD62-L + cells. CAR T cells manufactured with heme (heme-CAR T cells) have significantly reduced terminally exhausted phenotypes (PD1 +/LAG3 +/TIM3 +) and increased enrichment of effector memory cells (CD62L -CD45RA -) in both CD4 + and CD8 + subsets. Inquiry of cellular energetics of heme supplemented CAR T cells reveals reduced reactive oxygen species (ROS) and increased mitochondrial membrane potential, consistent with previous reports showing increased membrane potential in effector T cells for greater production of cytokines such as IL-17A, IL-17F, and IFNγ. In co-culture with tumor cells in vitro, heme-CAR T cells have higher tumor-specific lysis and production of TNFα and IL-2. CAR T-tumor cell engagement in the presence of heme resulted in significantly decreased proportions of exhausted CAR T cells and increased production of TNFα. Heme also increases BCMA surface expression in several MM cell lines, highlighting a viable strategy for enhancing BCMA CAR T engagement with MM. Lastly, MM patient bone marrow-localized T cells demonstrate significantly decreased terminally exhausted phenotypes and increased differentiation into effector subsets when expanded with heme. Conclusion: We show that heme increases effector T and CAR T cell function and decreases T cell exhaustion. Increased cytotoxicity of heme-supplemented CAR T cells was associated with reduced ROS and increased membrane potential. Ongoing in vivo studies aim to characterize the efficacy of heme-supplemented CAR T cells in MM xenograft models. Our studies have broader implications for investigating heme supplementation in ex vivo CAR T manufacturing for a wide range of hematological cancers and underscore the need for further investigation of heme uptake and metabolism for strategic enhancement of T-cell based immunotherapies.

Safety and Efficacy of GPRC5D CAR T Cell Therapy in Relapsed/Refractory Multiple Myeloma Patients

B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapies have shown breakthrough therapeutic effects in treating relapsed/refractory (R/R) multiple myeloma patients. However, relapses, including some that are BCMA negative, remain a challenge. Here we have developed a second-generation GPRC5D CAR-T, composed of a GPRC5D-binding scFv (derived from a humanized rabbit monoclonal antibody), a 4-1BB costimulatory domain, and a CD3ζ signaling domain. Following pre-clinical studies, we initiated a phase 1 dose-escalation clinical trial. Lymphodepletion with fludarabine and cyclophosphamide was performed before administering 3E6, 6E6, or 1E7/kg anti-GPRC5D CAR T cells. To date, we have enrolled a total of 7 R/R MM patients, including 3 patients with prior BCMA CAR-T treatment. All patients received autologous CAR T-cell infusions and were clinically evaluated between February 7th, 2023, and June 26th, 2023. No dose-limiting toxicities (DLT) were observed, even at the highest dose of 1E7/kg. Grade 3 or higher adverse events (AEs) were mainly hematological toxicities, such as neutropenia (6 [85.7%]), anemia (3 [43%]), and thrombocytopenia (3 [43%]). Cytokine release syndrome occurred in 6 (85.7%) of 7 patients, with all cases classified as grade 1 or 2. Notably, no neuro-toxicities were observed in any of the seven patients. The overall response rate was 85.7% (6/7 patients), comprising 3 complete responses or better, 3 partial responses, and one no-response (NR). The NR patient had previously shown no response to BCMA CAR-T therapy, with FACS analysis confirming the absence of both BCMA and GPRC5D on their tumor cells. Interestingly, the other two patients previously treated with BCMA CAR T-cell therapy, one reached complete response (CR), and the other reached partial response (PR). Remarkably, one patient diagnosed as COVID-19 positive two days before CAR-T infusion still received the CAR-T cell product, which led to a dramatic in vivo expansion of CAR-T cells, resulting in CR within one month after cell infusion. In conclusion, GPRC5D CAR T-cell therapy demonstrated promising clinical efficacy and tolerable safety in patients with R/R MM. For those who relapsed after anti-BCMA CAR T-cell therapy, GPRC5D CAR T-cell therapy offered an effective option (NCT05739188).“

Anti-TACI single and dual-targeting CAR T cells overcome BCMA antigen loss in multiple myeloma

Chimeric Antigen Receptor (CAR) T cells directed to B cell maturation antigen (BCMA) mediate profound responses in patients with multiple myeloma, but most patients do not achieve long-term complete remissions. In addition, recent evidence suggests that high-affinity binding to BCMA can result in on-target, off-tumor activity in the basal ganglia and can lead to fatal Parkinsonian-like disease. Here we develop CAR T cells against multiple myeloma using a binder to targeting transmembrane activator and CAML interactor (TACI) in mono and dual-specific formats with anti-BCMA. These CARs have robust, antigen-specific activity in vitro and in vivo. We also show that TACI RNA expression is limited in the basal ganglia, which may circumvent some of the toxicities recently reported with BCMA CARs. Thus, single-targeting TACI CARs may have a safer toxicity profile, whereas dual-specific BCMA-TACI CAR T cells have potential to avoid the antigen escape that can occur with single-antigen targeting.

Respiratory infections predominate after day 100 following B-cell maturation antigen–directed CAR T-cell therapy

AbstractInfections are an important complication after B-cell maturation antigen (BCMA)–directed chimeric antigen receptor (CAR) T-cell therapy and risks may differ between the early and late periods. We evaluated infections in 99 adults who received a first BCMA–directed CAR T-cell therapy (commercial and investigational autologous BCMA CAR T-cell products at the recommended phase 2 dose) for relapsed/refractory multiple myeloma between November 2016 and May 2022. Infections were recorded until day 365, if patients experienced symptoms with a microbiologic diagnosis, or for symptomatic site-specific infections treated with antimicrobials. One-year cumulative incidence functions were calculated based on time to first respiratory infection using dates of infection-free death and receipt of additional antineoplastic therapies as competing risks. Secondary analysis evaluated risk factors for late respiratory infections using univariate and multivariable Cox regression models. Thirty-seven patients (37%) experienced 64 infectious events over the first year after BCMA–directed CAR T-cell therapy, with 42 early infectious events (days, 0-100), and 22 late infectious events (days, 101-365). Respiratory infections were the most common site-specific infection and the relative proportion of respiratory infections increased in the late period (31% of early events vs 77% of late events). On multivariable analysis, hypogammaglobulinemia (hazard ratio [HR], 6.06; P = .044) and diagnosis of an early respiratory viral infection (HR, 2.95; P = .048) were independent risk factors for late respiratory infection. Respiratory infections predominate after BCMA CAR T-cell therapy, particularly after day 100. Hypogammaglobulinemia and diagnosis of an early respiratory infection are risk factors for late respiratory infections that may be used to guide targeted preventive strategies.

γ-Secretase inhibitor in combination with BCMA chimeric antigen receptor T-cell immunotherapy for individuals with relapsed or refractory multiple myeloma: a phase 1, first-in-human trial

γ-Secretase inhibitors (GSIs) increase B cell maturation antigen (BCMA) density on malignant plasma cells and enhance antitumour activity of BCMA chimeric antigen receptor (CAR) T cells in preclinical models. We aimed to evaluate the safety and identify the recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat (LY3039478) for individuals with relapsed or refractory multiple myeloma. We conducted a phase 1, first-in-human trial combining crenigacestat with BCMA CAR T-cells at a single cancer centre in Seattle, WA, USA. We included individuals aged 21 years or older with relapsed or refractory multiple myeloma, previous autologous stem-cell transplant or persistent disease after more than four cycles of induction therapy, and Eastern Cooperative Oncology Group performance status of 0-2, regardless of previous BCMA-targeted therapy. To assess the effect of the GSI on BCMA surface density on bone marrow plasma cells, participants received GSI during a pretreatment run-in, consisting of three doses administered 48 h apart. BCMA CAR T cells were infused at doses of 50 × 106 CAR T cells, 150 × 106 CAR T cells, 300 × 106 CAR T cells, and 450 × 106 CAR T cells (total cell dose), in combination with the 25 mg crenigacestat dosed three times a week for up to nine doses. The primary endpoints were the safety and recommended phase 2 dose of BCMA CAR T cells in combination with crenigacestat, an oral GSI. This study is registered with ClinicalTrials.gov, NCT03502577, and has met accrual goals. 19 participants were enrolled between June 1, 2018, and March 1, 2021, and one participant did not proceed with BCMA CAR T-cell infusion. 18 participants (eight [44%] men and ten [56%] women) with multiple myeloma received treatment between July 11, 2018, and April 14, 2021, with a median follow up of 36 months (95% CI 26 to not reached). The most common non-haematological adverse events of grade 3 or higher were hypophosphataemia in 14 (78%) participants, fatigue in 11 (61%), hypocalcaemia in nine (50%), and hypertension in seven (39%). Two deaths reported outside of the 28-day adverse event collection window were related to treatment. Participants were treated at doses up to 450 × 106 CAR+ cells, and the recommended phase 2 dose was not reached. Combining a GSI with BCMA CAR T cells appears to be well tolerated, and crenigacestat increases target antigen density. Deep responses were observed among heavily pretreated participants with multiple myeloma who had previously received BCMA-targeted therapy and those who were naive to previous BCMA-targeted therapy. Further study of GSIs given with BCMA-targeted therapeutics is warranted in clinical trials. Juno Therapeutics-a Bristol Myers Squibb company and the National Institutes of Health.

Limited efficacy of APRIL CAR in patients with multiple myeloma indicate challenges in the use of natural ligands for CAR T-cell therapy

BackgroundWe used a proliferating ligand (APRIL) to construct a ligand-based third generation chimeric antigen receptor (CAR) able to target two myeloma antigens, B-cell maturation antigen (BCMA) and transmembrane activator and...

Safety and Efficacy of BCMA-Targeted CAR-T Therapy in Geriatric Patients with Multiple Myeloma

Introduction: Risks of B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR-T) therapy for patients with multiple myeloma (MM) include cytokine release syndrome (CRS), immune effector-cell associated neurotoxicity syndrome (ICANS), and infections. Geriatric patients with MM are at higher risk of infections (Blimark 2015), and pre-existing comorbidities may complicate CRS and ICANS in older patients. While progression-free survival (PFS) was similar in older vs younger patients in the KarMMa trial of idecabtagene vicleucel (Berdeja 2020), BCMA CAR-T outcomes in the geriatric setting - including the risks of prolonged cytopenias, hypogammaglobulinemia, and infections - have not yet been analyzed. Methods: We analyzed all patients with MM who received any autologous BCMA CAR-T therapy from January 2017 - June 2022 at our institution. Key endpoints included CRS and ICANS incidence (American Society for Transplantation and Cellular Therapy criteria), days to absolute neutrophil count (ANC) recovery, incidence of hypogammaglobulinemia (IgG < 400 mg/dL), confirmed infections within 30 days of CAR-T infusion, response rates, time to measurable residual disease (MRD) negativity, PFS, and overall survival (OS). Characteristics were compared between older patients (age ≥70 at infusion) and younger patients using Fisher's & Kruskal-Wallis tests as appropriate. PFS and OS were compared using log-rank tests. Results: Of 77 analyzed patients (age range 33-77), 19 (25%) were aged ≥70. Gender, weight, performance status, years since diagnosis, presence of extramedullary disease, bone marrow plasma cell burden, high-risk cytogenetics, prior lines of therapy, and triple-class refractory status were similar between older and younger patients. Despite similar weights, older patients had lower Cockcroft-Gault creatinine clearances (median 69.2 vs 92.5 mL/min, p<0.001) and were more likely to receive reduced-dose fludarabine as part of lymphodepletion (53% vs 17%, p<0.01). Rates of any-grade CRS (79% vs 79%), rates of any-grade ICANS (11% vs 10%), and median days to ANC recovery (14.0 vs 13.5) were similar between older and younger patients. Rates of baseline hypogammaglobulinemia were 32% in older patients vs 28% in younger patients (p=0.77), while post-infusion hypogammaglobulinemia occurred in 84% vs 71%, respectively (p=0.36). Six infections occurred before Day +30, all in the younger cohort: rhinovirus (n=2), Staphylococcus epidermidis (n=2), CMV reactivation (n=1), and COVID-19 (n=1). There were two deaths before Day +100, both due to progressive MM in younger patients. Overall response rates (84% vs 90%) were similar between older and younger patients. Median PFS (Figure 1A) was not reached (NR) in older patients (95% CI 10.6-NR) vs 13.1 months in younger patients (95% CI 9.4-33.1), with p=0.19. Median OS (Figure 1B) was NR in older patients (95% CI NR-NR) vs 30.0 months (95% CI 24.8-NR) in younger patients, with p=0.05. Two-year survival probabilities were 93% vs 66%, respectively. Discussion: While limited by small sample size and unmeasured confounders, our retrospective analysis did not demonstrate significant increases in CAR-T toxicity among patients aged ≥70. Differences in rates of hypogammaglobulinemia between older and younger patients did not reach statistical significance. Fludarabine dose reductions during lymphodepletion (as expected with age-adjusted creatinine clearances) did not impair CAR-T efficacy among geriatric patients. Older patients paradoxically had a significantly better OS compared to younger patients, which may have been due to selection bias in favor of disproportionately healthy geriatric patients with regard to CAR-T candidacy. Overall, our data suggest that BCMA CAR-T is a safe and effective option for older patients with MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interim Phase I Clinical Data of FT576 As Monotherapy and in Combination with Daratumumab in Subjects with Relapsed/Refractory Multiple Myeloma

Introduction: Despite the recent FDA approval of autologous chimeric antigen receptor (CAR) T-cell therapies targeting B-cell maturation antigen (BCMA), multiple myeloma (MM) remains incurable and relapsed/refractory (R/R) disease remains an area of high unmet medical need. Additionally, patient access to autologous CAR T-cell therapies is currently limited due to manufacturing constraints, the need for bridging therapy, and potentially life-threatening toxicities including cytokine release syndrome and neurologic toxicities (Munshi et al. 2021). Off-the-shelf natural killer (NK) cell therapies may offer an improved therapeutic profile and broader patient access than autologous CAR T-cell therapies. FT576 is a first-of-kind, multiplexed-engineered BCMA CAR NK cell therapy generated from a clonal master engineered induced pluripotent stem cell line, which can be used as a renewable source for the mass production of off-the-shelf NK cells of uniform composition. FT576 is engineered with 4 modalities to combine multifaceted innate immunity with multi-antigen-targeting capability: (1) high-affinity 158V, non-cleavable CD16 (hnCD16) Fc receptor for augmented antibody-dependent cellular cytotoxicity; (2) IL-15/IL-15 receptor fusion that promotes NK cell persistence; (3) CD38 knockout to mitigate NK cell fratricide by CD38-directed monoclonal antibodies (mAbs) and that provides improved metabolic fitness and resistance to oxidative stress within the tumor microenvironment; and (4) a BCMA-directed CAR to target plasma cells. These modalities are designed to enhance the potency and persistence of FT576 and to enable multi-antigen targeting when combined with tumor-targeting mAbs. In preclinical studies, FT576 combined with the anti-CD38 mAb daratumumab demonstrated highly effective tumor control compared to either treatment alone or to primary CAR T cells in a disseminated MM xenograft model (Goodridge et al. 2021), suggestive that limitations in MM treatment confounded by clonal heterogeneity and antigen loss can be overcome with a dual antigen-targeting approach. Methods: This is a Phase I trial of FT576 in patients with R/R MM. The primary objectives are to assess safety and tolerability and to determine the recommended Phase II dose of FT576, given as single or multiple doses, as monotherapy and in combination with daratumumab in R/R MM. Key secondary objectives include anti-tumor activity and pharmacokinetics. The dose-escalation stage of the trial has the following 4 arms: single-dose FT576 monotherapy on Day 1 (Regimen A); multi-dose FT576 monotherapy on Days 1 and 15 (Regimen A1); single-dose FT576 + daratumumab on Day 1 (Regimen B); and multi-dose FT576 + daratumumab on Days 1 and 15 (Regimen B1). Dose levels of FT576 starting from 100 million cells/dose are being evaluated using a modified toxicity probability interval dose-escalation design. Daratumumab is administered per approved dose and schedule. Outpatient conditioning chemotherapy consists of 3 consecutive days of fludarabine and cyclophosphamide administered prior to the first dose of FT576. Results: As of a data cutoff date of 18 Jul 2022, 9 patients with R/R MM were treated and evaluable for safety and efficacy, in the first 2 dose levels of Regimen A (n = 6) and in the first dose level of Regimen B (n = 3). No dose-limiting toxicities, and no events of any grade of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome, or graft-versus-host disease (GvHD), were observed. Conclusions: Administration of a single dose of FT576 at 100 or 300 million cells/dose alone or in combination with daratumumab is safe and well tolerated thus far without CRS, neurotoxicity, or GvHD. Interim clinical data, including safety and tolerability and initial anti-tumor activity, from the ongoing Phase I dose-escalation study of FT576 will be presented at the conference.

A Preliminary Study on the Safety and Efficacy of a Novel Human BCMA-Targeting CAR-T Therapy in Patients with Relapsed/Refractory Multiple Myeloma

IntroductionMultiple myeloma is a hematological malignancy that is prone to be companied by bone marrow destruction, renal impairment and extramedullary infiltration. Current treatments include proteasome inhibitor, immunomodulatory drug, hematopoietic stem cell transplantation, and monoclonal antibody targeted therapies. However, it is still clinically incurable. Chimeric antigen receptor (CAR) T-cell therapy is a new immune targeted therapeutic strategy. It is reported better clinical efficacy for relapsed/refractory multiple myeloma (r/r MM) treatment has been achieved by immunotherapies targeted B-cell maturation antigen (BCMA). Therefore, it is important to investigate the treatment of a novel human BCMA-specific CAR-T therapy for r/r MM.ObjectiveThe objective of this clinical study is to evaluate the safety and efficacy of the novel human BCMA-targeting CAR-T therapy in patients with r/r MM, especially patients who relapsed from prior CAR-T therapy.MethodThis work is a clinical study registered and investigated by our center. CD3+ T cells were negatively selected from patients' peripheral blood mononuclear cells, activated and modified by lentivirus to produce anti-BCMA CAR-T cells. The cells were administrated intravenously to patients after expanding for 7-13 days in vitro. 24 patients had enrolled in this study, with 13 males and 11 females. The median age was 53 years old (range,41-75), and patients with cytogenetically high risk factors accounting for 41.66% (10/24). 50% (12/24) was infiltrated with extramedullary lesions. 16.6% (4/24) of them had relapsed from other CAR-T therapies before this enrollment. 50% (12/24) had been previously conducted autologous hematopoietic stem cell transplantation (HSCT), whereas 4.16% (1/24) with allogeneic HSCT. Patients with the expression of BCMA in the plasma cells higher than 30%, accounted for 25% (6/24). 2-3 days after being administered the lymphodepleting chemotherapy regimen, CAR-T cells were infused intravenously. The indicators of patients' condition were detected, including inflammatory cytokine concentration, serum protein levels, CAR-T cell number copies, and the proportion of plasma cells by bone marrow biopsy. The improvement of patients, the occurrence of adverse reactions, the incidence and grade of cytokine release syndrome (CRS), was analyzed and evaluated.ResultAll patients received infusions of CAR-T positive cells at the average dose of 9.45×10 6/kg (5-17.5) and the median injection day is the 10th day (8th-13th day) after cell isolation. After infusion, 100% (24/24) of the patients had fever lasting for 48 hours, with 37.5% (9/24) of them showing low blood pressure and being treated with drug. Heart rate increase was found in 45.8% (11/24). Nausea, diarrhea and transient consciousness disorder occurred in 50% (12/24), 33.3% (8/24), and 12.5% (3/24) of them, respectively. 16.6% (4/24) was administrated with dexamethasone to relieve symptoms, with the total dose less than 20 mg, while nobody was treated with IL-6 receptor antagonist. CAR-T cells had expanded in all patients, reaching the peak at the 4th day after infusion (Figure). The levels of IL-6, IL-8 and IFN-γ in peripheral blood also increased significantly. The incidence of CRS is 100%, of which grade I, II and III is 62.5%, 33.3% and 4.2%, respectively. 2 patients showed grade I CRES, constituting 8.3% (2/24). All patients were assessed for the efficacy of CAR-T cells 2 weeks after infusion. ORR was 100%, with 4.2% (1/24) MR, 8.3% (2/24) PR, 62.5% (15/24) VGPR and 25% (6/24) CR. 18 patients were treated for more than 1 month, with 11.1% (2/18) PR, 44.4% (8/18) VGPR, 11.1% (2/18) CR, and 33.3% (6/18) sCR. 16 patients were infused before more than 2 months, with 25% (4/16) VGPR, 12.5% (2/16) CR, 50% (8/16) sCR, and 12.5% (2/16) PD. 6 patents were administrated more than 3 months ago, with 1 developing deep remission to sCR from VGPR. The others remain the same condition.ConclusionThe novel human BCMA targeted CAR-T cell therapy of this study showed safety and efficacy in the treatment of r/r MM patients with extramedullary infiltration, high-risk cytogenetical factors as well as relapse with prior BCMA CAR exposures. Deep remission can be achieved. However, more observation need to be conducted. The CAR-T treatment of BCMA target still cannot prevent the disease progress of a small numbers of patients. The control after CAR-T therapy needs more investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Infectious complications of CAR T-cell therapy across novel antigen targets in the first 30 days

AbstractInfections are a known complication of chimeric antigen receptor (CAR) T-cell therapy with data largely emerging from CD19 CAR T-cell targeting. As CAR T-cell therapy continues to evolve, infection risks and management thereof will become increasingly important to optimize outcomes across the spectrum of antigens and disease targeted. We retrospectively characterized infectious complications occurring in 162 children and adults treated among 5 phase 1 CAR T-cell clinical trials. Trials included targeting of CD19, CD22, disialoganglioside (GD2) or B-cell maturation antigen (BCMA). Fifty-three patients (32.7%) had 76 infections between lymphocyte depleting (LD) chemotherapy and day 30 (D30); with the majority of infections (61, 80.3%) occurring between day 0 (D0) and D30. By trial, the highest proportion of infections was seen with CD22 CAR T cells (n = 23/53; 43.4%), followed by BCMA CAR T cells (n = 9/24; 37.5%). By disease, patients with multiple myeloma had the highest proportion of infections (9/24; 37.5%) followed by acute lymphoblastic leukemia (36/102; 35.3%). Grade 4 infections were rare (n = 4; 2.5%). Between D0 and D30, bacteremia and bacterial site infections were the most common infection type. In univariate analysis, increasing prior lines of therapy, recent infection within 100 days of LD chemotherapy, corticosteroid or tocilizumab use, and fever and neutropenia were associated with a higher risk of infection. In a multivariable analysis, only prior lines of therapy and recent infection were associated with higher risk of infection. In conclusion, we provide a broad overview of infection risk within the first 30 days post infusion across a host of multiple targets and diseases, elucidating both unique characteristics and commonalities highlighting aspects important to improving patient outcomes.

ATRA Augments BCMA Expression on Myeloma Cells and Enhances Recognition By BCMA-CAR T-Cells

Background: B cell maturation antigen (BCMA) is a B-lineage antigen that is retained on malignant plasma cells in multiple myeloma (MM), and is under investigation as a target antigen for humoral and cellular immunotherapy. Targeting BCMA with chimeric antigen receptor (CAR) T-cells, T-cell engaging antibodies and antibody-drug conjugates has resulted in high rates of clinical responses however, the depth and durability of these responses is still not satisfactory and most patients ultimately relapse. This has been attributed at least in part to low or non-uniform BCMA expression on MM cells, as well as MM cell escape after BCMA down-regulation or even loss. Here, we show that epigenetic modulation with all-trans retinoic acid (ATRA) augments BCMA expression at the gene (and protein) level and leads to enhanced BCMA molecule density on the surface of MM cells that translates into increased anti-MM potency of BCMA CAR T-cells. Methods: Primary MM cells and myeloma cell lines were treated with titrated doses of ATRA (25, 50, 100 nM), alone and in combination with the g-secretase inhibitor crenigacestat (10 nM). BCMA expression was analyzed by flow cytometry, RT-qPCR and direct stochastic optical reconstruction microscopy (dSTORM). BCMA CAR T-cells were derived from healthy donors and MM patients (n&amp;gt;6) and their anti-MM function analyzed in vitro and in the NSG/MM.1S murine xenograft model in vivo. Results: By RT-qPCR, we observed a 1.8-fold (MM.1S) and 2.1-fold (OPM-2) increase in BCMA gene expression after treatment with 50 nM ATRA for 72 hours. By flow-cytometry, we confirmed increased BCMA protein expression, with 1.9-fold (MM.1S and OPM-2) increase in mean fluorescence intensity relative to isotype control staining. Super-resolution dSTORM microscopy on MM.1S cells confirmed the increase in BCMA protein expression and showed a homogenous distribution pattern of BCMA molecules across the cell surface without an increase in cluster formation. These data were confirmed with primary MM cells from patients with newly diagnosed (n=7) and relapsed/refractory (n=11) MM. The increase in MFI for BCMA expression on primary MM cells after ATRA treatment was 1.2-fold - 2.2-fold (mean: 1.6-fold; p=.01 at 50 nM ATRA). By ELISA, we did not detect increased levels of soluble BCMA protein in supernatant of MM.1S cells after ATRA treatment. Accordingly, we found superior cytolytic activity, cytokine secretion and proliferation of CD8+ and CD4+BCMA CAR T-cells in response to ATRA-treated vs. non-treated primary MM cells and MM cell lines. In the NSG/MM.1S xenograft model, we confirmed increased BCMA expression on MM.1S after systemic treatment with ATRA, and superior anti-MM activity after adoptive transfer of BCMA CAR T-cells. Further, we confirmed that epigenetic modulation of BCMA-expression with ATRA works synergistically with g-secretase inhibitor treatment that has recently been shown to prevent cleavage of BCMA molecules from the surface of MM cells (Pont Blood 2019). Combination treatment with ATRA and the g-secretase inhibitor crenigacestat led to higher BCMA density on primary MM cells (and cell lines) than each single-agent treatment alone, resulting in maximum reactivity of by BCMA CAR T-cells in vitro and in vivo. Conclusions: Taken together, the data show that BCMA expression on MM cells can be increased by epigenetic modulation with ATRA. After ATRA treatment, MM cells have increased susceptibility to BCMA CAR T-cell treatment in pre-clinical models vitro and in vivo, that can be increased even further by combination treatment of ATRA and g-secretase inhibitors. These data suggest the potential to improve responses (depth and durability) of immunotherapies directed against BCMA. Disclosures Einsele: Takeda: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau.

Systematically optimized BCMA/CS1 bispecific CAR-T cells robustly control heterogeneous multiple myeloma

Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy against B-cell malignancies, yet marked vulnerability to antigen escape and tumor relapse exists. Here we report the rational design and optimization of bispecific CAR-T cells with robust activity against heterogeneous multiple myeloma (MM) that is resistant to conventional CAR-T cell therapy targeting B-cell maturation antigen (BCMA). We demonstrate that BCMA/CS1 bispecific CAR-T cells exhibit superior CAR expression and function compared to T cells that co-express individual BCMA and CS1 CARs. Combination therapy with anti–PD-1 antibody further accelerates the rate of initial tumor clearance in vivo, while CAR-T cell treatment alone achieves durable tumor-free survival even upon tumor re-challenge. Taken together, the BCMA/CS1 bispecific CAR presents a promising treatment approach to prevent antigen escape in CAR-T cell therapy against MM, and the vertically integrated optimization process can be used to develop robust cell-based therapy against novel disease targets.

Bispecific CAR T cells for multiple myeloma: natural ligand compared to tandem scFv design

Abstract One of the most promising candidates for Chimeric Antigen Receptor (CAR) T cell therapy beyond CAR19 is treatment of multiple myeloma with CAR T cells targeting B cell maturation antigen (BCMA). However, current reports of BCMA CAR in the clinic have a median progression free survival of 11.8 months, suggesting that targeting BCMA alone may not be sufficient. Evidence suggests patients treated with BCMA-targeted therapies may be thwarted by BCMA negative relapse. We have developed a novel second generation CAR T cell against another multiple myeloma target: transmembrane activator and CAML interactor (TACI). Mice were immunized against TACI and we designed a new scFv targeting TACI based on the resulting antibodies. Anti-TACI CAR T cells are cytotoxic in vitro and in vivo against multiple myeloma. To overcome single antigen loss, we designed bispecific tandem scFv CAR T cells targeting BCMA and TACI. These two antigens share a natural ligand, A Proliferation-Inducing Ligand (APRIL), which has also been used to design a dual-targeting CAR called TriPRIL. We show that these dual targeting CARs, based on tandem scFv or natural ligand design, have similar efficacy against wild type multiple myeloma models. However, this changes in the context of single antigen loss. We have characterized the proliferative (population doublings) and activation capability (CD69) of these CARs, as well as their memory (CCR7, CD45RA) and exhaustion phenotype (PD-1, TIM-3, LAG-3), with long term exposure to single antigen. Our studies show that sensitivity to antigen density differs between the tandem bispecifics and the natural ligand CAR. These data provide insight into how structural differences between dual-targeting CAR T cells affects their function.

ALLO-715, an Allogeneic BCMA CAR T Therapy Possessing an Off-Switch for the Treatment of Multiple Myeloma

Autologous chimeric antigen receptor (CAR) T cells targeting B-Cell Maturation Antigen (BCMA) have demonstrated promising clinical activity, inducing durable responses in patients with relapsed/refractory multiple myeloma (MM). Development of autologous CAR T therapies is however limited by logistical challenges and the time required for manufacturing, which has to be done for each patient. In addition, manufacturing may not be feasible in some patients. An allogeneic approach that utilizes engineered cells from a healthy donor could potentially expand patient access to these therapies by providing a readily available off-the-shelf product. We have previously described the screening of a library of single chain variable fragments (scFvs) with high affinity to human BCMA and the identification of candidate BCMA CARs with potent antitumor activity. Here we sought to further characterize ALLO-715, our lead allogeneic BCMA CAR T cell product, for its specificity to human BCMA, antitumor efficacy in vitro using a long-term killing assay and in xenograft mouse models with physiologic levels of human IL-7 and IL-15, and suitability for scale-up manufacturing.Allogeneic ALLO-715 CAR T cells were generated by lentiviral transduction with a second generation CAR construct incorporating a novel scFv derived from a fully-human antibody with high affinity to BCMA (KD value ~ 5 nM, determined at 37°C) and featuring a rituximab-driven off-switch. Transduced T cells were then transfected with mRNAs encoding Transcription Activator-Like Effector Nucleases (TALEN®) designed to specifically disrupt the T cell receptor alpha chain and CD52 loci. These modifications result in a cell product with a lower risk of TCR-mediated graft-versus-host disease and resistance to the CD52 antibody alemtuzumab, a lymphodepleting agent. BCMA CAR T cells exhibited robust cell expansion, with low levels of tonic signaling that resulted in minimal differentiation (> 50% Tscm/Tcm phenotype). In in vitro assays, ALLO-715 CAR T cells displayed potent cytotoxic activity when co-cultured with the target cell lines MM.1S, Molp-8, and BCMA-REH but negligible cytotoxicity against BCMA-negative REH cells.The high proliferative potential indicated by the high frequency of memory T cells was validated in long-term killing assays, where ALLO-715 CAR T cells showed substantial expansion in the presence of MM.1S cells with no evidence of exhaustion or diminished cytolytic activity after seven days of continuous exposure to target. The potency of ALLO-715 CAR T cells was unaffected by high concentrations of soluble BCMA (>10 ug/mL), which has been shown previously to interfere with the activity of some BCMA-specific CARs.In MM xenograft mouse models, ALLO-715 CAR T cells were highly efficacious at single dose. High serum IL-15 levels have been associated with CAR T cell expansion in clinical trials. To evaluate the impact of homeostatic cytokines on CAR T cell survival and antitumor activity in our xenograft models, mice were administered adeno-associated viruses (AAV) for the expression of human IL-7 and IL-15. In the presence of physiological concentrations of these cytokines, enhanced BCMA CAR T cell expansion and anti-tumor activity were observed.To assess potential off-target interactions of ALLO-715 CAR, tissue cross-reactivity studies were carried out on standard human tissue panels using a scFv-human IgG fusion protein. Consistent with the limited expression pattern of BCMA, reactivity was seen on scattered cells in lymphoid tissues such as tonsil and abundantly on BCMA-expressing cell lines, but no appreciable staining was detected in other tissues.We examined BCMA CAR T cells manufactured following a proprietary GMP-like clinical scale process and found that cell expansion and viability, T cell phenotype and in vivo antitumor efficacy were preserved.These results demonstrate the potential of ALLO-715 as a novel allogeneic BCMA CAR T therapy for the treatment of relapsed/refractory MM and other BCMA-positive malignancies. DisclosuresSommer:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boldajipour:Pfizer Inc.: Employment, Patents & Royalties. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Galetto:Cellectis SA: Employment, Equity Ownership, Patents & Royalties. Bentley:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Ni:Allogene Therapeutics: Employment, Equity Ownership. Leonard:Allogene Therapeutics: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Fully Human Bcma Targeted Chimeric Antigen Receptor T Cells Administered in a Defined Composition Demonstrate Potency at Low Doses in Advanced Stage High Risk Multiple Myeloma

Fully Human Bcma Targeted Chimeric Antigen Receptor T Cells Administered in a Defined Composition Demonstrate Potency at Low Doses in Advanced Stage High Risk Multiple Myeloma