anti-BCMA Chimeric Antigen Receptor Research Articles

Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment of relapsed/refractory multiple myeloma (RRMM). Robust variables that predict long-term response are currently missing. Limited data are especially available on the impact of bridging therapies on manufacturing and outcome. We conducted a longitudinal single-cell multi-omics study to identify factors that predict response to BCMA-directed CAR T cells. Changes in the immune microenvironment associated with response were analyzed as well as the impact of prior bridging therapy with bispecific antibodies on subsequent CAR T cell manufacturing and outcome. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 29 consecutive MM patients treated with commercially available anti-BCMA CAR T cells on the day of leukapheresis as well as days 30 and 100 after CAR T cell infusion. PBMCs were subjected to single cell RNA, T-cell receptor (TCR) and B-cell receptor (BCR) sequencing. A custom panel of 57 oligonucleotide-coupled antibodies was used to study surface proteomics. Downstream analyses were performed with Seurat. Differences in cellular compositions at all three time points were analyzed with scCODA. To analyze CAR T cell functionality, CAR T cells from peripheral blood were isolated 7 days after infusion and subjected to an in vitro cytotoxicity assay after expansion and stimulation. Patients were grouped based on their best response following CAR T cell infusion (CR: n=12, non CR: n=17). Results: In total, 375,338 cells were sequenced (median 7246 cells/sample, range 1,569-10,972 cells) and 354,878 cells (94.5%) passed quality assessment. Quantitative and qualitative differences in the cellular composition of peripheral blood between CR and non CR patients were detected at the time of leukapheresis as well as on days 30 and 100 following infusion. CR patients harbored significantly more CD8+ effector memory T cells (TEM) at leukapheresis and less NK cells on day 30 after therapy compared to non CR patients. Regulatory T cells isolated at the time of leukapheresis from non CR patients exhibited significantly higher surface protein levels of CXCR3, CD40, CD95 and KLRG1 (p<0.015, respectively) that have been associated with T cell senescence and impaired tumor immunity. No significant differences in cell numbers between CR and non CR were detected on day 100 after CAR T cell infusion. However, single cell TCR analysis revealed an increasing diversity in the TCR repertoire over time in patients with CR, while Shannon diversity decreased from leukapheresis over day 30 to day 100 in non CR patients (p=0.004). The prior administration of the bispecific antibody teclistamab had no significant impact on the quantitative cellular composition at the time of leukapheresis. However, termination of manufacturing in the first attempt occurred in all patients with a close proximity of teclistimab administration and apheresis. Differential gene expression analysis showed that the application of teclistamab was associated with impaired T cell activation and exhaustion indicated by upregulation of e.g. CTLA4, TIGIT, LAG3 and GZMK. After discontinuation of teclistamab (median 4 weeks) and successful manufacturing of CAR T cells, we found no significant differences for in vitro cytotoxicity and in vivo expansion of CAR T cells. CAR T cells isolated at day 7 post-infusion from patients in CR, non CR or with prior teclistamab exposure, effectively eliminated MM cells (U-266). Tracking of single CAR T cells over time showed that the majority of CAR+ cells were CD8+ TEMs regardless of remission achievement or prior teclistamab exposure. Conclusion: We demonstrate that differences between MM patients achieving a CR and patients with suboptimal response upon anti-BCMA CAR T cell therapy can already be identified at the time of leukapheresis. Long-term changes associated with CR include a diversification of the TCR repertoire. Successful CAR T cell manufacturing is hampered by exposure to bispecific antibodies but can be successfully achieved by allowing for a wash-out phase of ca. 4 weeks.

In Vivo Anti-BCMA CAR T-Cell Expansion Kinetics Correlate with Early IMWG Response in RRMM: A Single Institution Study with Comparative Analysis of Idecabtagene Vicleucel and Ciltacabtagene Autoleucel

Impact of Gamma-Secretase Inhibition on the Multiple Myeloma Immune Microenvironment

Safety and Efficacy of a Locally Produced Novel Anti-BCMA Chimeric Antigen Receptor T-Cell (CART) (HBI0101) for the Treatment of Relapsed and Refractory Multiple Myeloma

The Phase 2 CARTITUDE-2 Trial: Updated Efficacy and Safety of Ciltacabtagene Autoleucel in Patients with Multiple Myeloma and 1-3 Prior Lines of Therapy (Cohort A) and with Early Relapse after First Line Treatment (Cohort B)

Anti-BCMA Dual Epitope-Binding CAR-Marrow Infiltrating Lymphocytes (MILs) Could Offer a Potent Innovative Immunotherapeutic Tool Against Multiple Myeloma

Incidence and Outcomes of Pneumocystis Pneumonia Following Chimeric Antigen Receptor T-Cell Therapy: A Real-World Analysis

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

Comparison of Fludarabine/Cyclophosphamide Vs Bendamustine As Lymphodepleting Regimen for CAR-T: A Safety and Efficacy Analysis in Patients with Relapsed/ Refractory Multiple Myeloma

Feasibility of a Novel Academic Anti-BCMA Chimeric Antigen Receptor T-Cell (CART) (HBI0101) for the Treatment of Relapsed and Refractory AL Amyloidosis

OA-34 Feasibility of a novel academic anti-BCMA chimeric antigen receptor T-cell (CART) (HBI0101) for the treatment of relapsed and refractory AL amyloidosis

P-026 Safety and efficacy of a locally produced novel anti-BCMA chimeric antigen receptor T-cell (CART) (HBI0101) for the treatment of relapsed and refractory multiple myeloma

B cell maturation antigen (BCMA) target loss is considered to be a rare event that mediates multiple myeloma (MM) resistance to anti-BCMA chimeric antigen receptor T cell (CAR T) or bispecific T cell engager (TCE) therapies. Emerging data report that downregulation of G-protein-coupled receptor family C group 5 member D (GPRC5D) protein often occurs at relapse after anti-GPRC5D CAR T therapy. To examine the tumor-intrinsic factors that promote MM antigen escape, we performed combined bulk and single-cell whole-genome sequencing and copy number variation analysis of 30 patients treated with anti-BCMA and/or anti-GPRC5D CAR T/TCE therapy. In two cases, MM relapse post-TCE/CAR T therapy was driven by BCMA-negative clones harboring focal biallelic deletions at the TNFRSF17 locus at relapse or by selective expansion of pre-existing subclones with biallelic TNFRSF17 loss. In another five cases of relapse, newly detected, nontruncating, missense mutations or in-frame deletions in the extracellular domain of BCMA negated the efficacies of anti-BCMA TCE therapies, despite detectable surface BCMA protein expression. In the present study, we also report four cases of MM relapse with biallelic mutations of GPRC5D after anti-GPRC5D TCE therapy, including two cases with convergent evolution where multiple subclones lost GPRC5D through somatic events. Immunoselection of BCMA- or GPRC5D-negative or mutant clones is an important tumor-intrinsic driver of relapse post-targeted therapies. Mutational events on BCMA confer distinct sensitivities toward different anti-BCMA therapies, underscoring the importance of considering the tumor antigen landscape for optimal design and selection of targeted immunotherapies in MM.

Most patients with multiple myeloma experience disease relapse after treatment with a B-cell maturation antigen-targeted therapy (BCMA-TT), and data describing outcomes for patients treated with sequential BCMA-TT are limited. We analyzed clinical outcomes for patients infused with standard-of-care idecabtagene vicleucel, an anti-BCMA chimeric antigen receptor (CAR) T-cell therapy, at 11 US medical centers. A total of 50 patients with prior BCMA-TT exposure (38 antibody-drug conjugate, 7 bispecific, 5 CAR T) and 153 patients with no prior BCMA-TT were infused with ide-cel, with a median follow-up duration of 4.5 and 6.0 months, respectively. Safety outcomes between cohorts were comparable. The prior BCMA-TT cohort had a lower overall response rate (74% versus 88%; p = 0.021), median duration of response (7.4 versus 9.6 months; p = 0.03), and median progression-free survival (3.2 months versus 9.0 months; p = 0.0002) compared to the cohort without prior BCMA-TT. All five patients who received a prior anti-BCMA CAR T responded to ide-cel, and survival outcomes were best for this subgroup. In conclusion, treatment with ide-cel yielded meaningful clinical responses in real-world patients exposed to a prior BCMA-TT, though response rates and durability were suboptimal compared to those not treated with a prior BCMA-TT.

γ-Secretase inhibitors plus anti-BCMA chimeric antigen receptor T cells

Introduction: Anti-BCMA chimeric antigen receptor (CAR) T-cell therapy showed excellent efficacy in patients with relapsed/refractory multiple myeloma (R/R MM). Point-of-care (POC) CAR manufacturing abrogates the need for cryopreservation and shipment of cells, thus shortening the manufacturing process and reducing the necessity of bridging therapy. We report outcomes of phase 1b/2 single-center clinical trial of autologous POC anti-BCMA CAR T-cell therapy in patients (pts) with R/R MM treated with ≥3 prior therapies (NCT05243212). Methods: Pts underwent a single peripheral blood leukapheresis. Fresh T-cells were transduced with retroviral vector encoding the anti-BCMA CAR (based on 11D5-3 ScFv, CD28 costimulatory domain, and CD3-ζ signaling domain). Cell dose was 6 × 106/kg (dose level I [n = 3]) or 9 × 106/kg (dose level II [n = 29]) CAR T-cells. Response was defined per IMWG criteria. Last follow-up was as of 02/2023. Results: All 32 pts (median age 60, IQR 54–67) enrolled received CAR T-cell infusion in a median of 11 days (IQR 11–11) after leukapheresis. Only 2 pts received bridging chemotherapy. The median number of prior therapies was 4 (IQR 3–5), with 59% and 34% of the pts being penta- and quad-refractory, respectively. 7 (22%) pts had prior exposure to BCMA-targeted therapy (belantamab mafodotin, n = 5 [16%]; talqeutamab, n = 2 [6%]). At enrollment, 10 (34%) pts had high-risk cytogenetics, 7 (22%) had double-hit myeloma, and 17 (57%) had extramedullary involvement. Only 17 (53%) and 2 (16%) pts were eligible to enroll in the KarMMa (NEJM, 2021) and CARTITUDE-1 (Lancet, 2021) studies, respectively. One patient (3%) developed grade 3 cytokine release syndrome. Immune effector cell-associated neurotoxicity syndrome was not noted. Grade 3–4 neutropenia and thrombocytopenia occurred in 31 (97%) and 16 (50%), respectively. Anemia requiring transfusion occurred in 14 (44%) pts. Cellular therapy-related death was not observed. The median follow-up was 3.9 months (IQR 2.6–7.3). Best Overall response rate (PR at least) was 59% (VGPR at least, 40%). Median time to first response was 31 days (95% CI: 26–33). Estimated 6-months OS, PFS and duration of response were 89% (95% CI: 75–100), 48% (95% CI: 33–72), and 63% (95% CI: 41–97), respectively. Patients with prior exposure to BCMA-targeted therapies had an inferior PFS (HR 3.4 [95% CI: 1.2–9.7] p = 0.03; Figure 1). Conclusion: POC anti-BCMA CAR T-cells induced high response rates with an excellent safety profile in high-risk MM patients mostly not eligible to be enrolled in the pivotal trials. The rapid CAR-T production time obviated the need for bridging therapy in most patients. It is noteworthy that prior exposure to BCMA-targeted therapies is associated with dismal PFS, hence those therapies should be carefully considered when CAR T-cell therapy might be intended. Encore Abstract—previously submitted to EHA 2023 Keywords: cellular therapies, multiple myeloma No conflicts of interests pertinent to the abstract. * H. Magen, E. Shkury and A. Avigdor contributed equally to the abstract.

Abstract Background: Multiple myeloma (MM) is an incurable disease with a heterogenous clinical course and genomic landscape. Autologous anti-BCMA chimeric antigen receptor (CAR) T-cells are a promising new therapy, but determinants of response and resistance are not well known. Cell-free DNA (cfDNA) is a useful tool to study MM as it allows for repeated, non-invasive tumor assessment. We apply a novel method for simultaneously tracking tumor mutations and CAR T-cells from cfDNA using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq). Methods: We designed a 480kb CAPP-Seq hybrid capture panel to identify mutations, track tumor burden and minimal residual disease, and detect cfDNA derived from the CAR transgene (CAR-cfDNA) in patients receiving idecabtagene vicleucel (ide-cel). Flow cytometry (FC) for enumeration of CAR T-cells was performed from peripheral blood mononuclear cells (PBMCs) when available. Results: We profiled 153 biologic samples, including plasma, PBMCs, and bone marrow mononuclear cells, from 15 patients receiving ide-cel and 18 healthy controls. We observed a median of 84 SNVs (range 30-277) prior to therapy. Patients with prolonged responses (>90 days) had significantly lower circulating tumor DNA (ctDNA) at day 28 post-infusion than patients with early progression (<90 days) (0.6 vs. 4.6 log haploid genome equivalents (hGE)/mL; p=0.002). Additionally, higher ctDNA at D28 was prognostic for time to progression (TTP) (HR=1.67, p=0.019). We validated CAR-cfDNA detection by comparison with FC from PBMCs at matched timepoints (n=38), finding a significant correlation (rho=0.79, p=3E-09). CAR-cfDNA typically reached its peak level around D14 (median 332 hGE/mL), with ctDNA declining at the same time-point. Thus, CAR-cfDNA levels and ctDNA burden were inversely correlated (rho= -0.3, p=0.019). Surprisingly, peak CAR expansion was not associated with TTP (HR=1, p=0.463). However, lower CAR-cfDNA at D28 was prognostic for inferior TTP (HR=2.68, p=0.011). This suggests CAR persistence may play a more important role in clinical outcomes. Furthermore, among progressors, time until loss of detectable CAR-cfDNA correlated with TTP (rho=0.81, p=0.02). Patients who progressed before day 90 had a median CAR persistence of 28 days. In contrast, patients who progressed after day 90 had a median of 137 days and often had emergent copy number alterations in ctDNA at relapse. This included one case with emergent loss of chr16, where TNFRSF17 (BCMA), resides. This event was detected 36 days prior to clinical relapse; BCMA loss was validated via whole-genome sequencing and immunohistochemistry staining of the tumor. Conclusions: Cell-free DNA is a promising biomarker for mutational genotyping, disease monitoring, and tracking CAR T-cells in MM. The persistence of CAR-cfDNA has particular prognostic importance and novel strategies to increase CAR persistence should be explored. Citation Format: Mia Carleton, Hitomi Hosoya, Kailee L. Tanaka, Brian Sworder, Vanna Hovanky, Bita Sahaf, Matthew J. Frank, George E. Duran, Tian Y. Zhang, Sally Arai, David Iberri, Michaela Liedtke, David B. Miklos, Michael S. Khodadoust, Surbhi Sidana, David M. Kurtz. Tumor and immune determinants of response to anti-BCMA CAR T-cell therapy in multiple myeloma using cell-free DNA. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5707.

Abstract Recent advances in treatment options for multiple myeloma (MM) have improved patient outcomes, however, MM remains incurable and options are limited for relapsed/refractory disease. Memory-like (ML) differentiation of NK cells using brief IL-12, IL-15, and IL-18 activation generates a safe allogenic NK cellular therapy for acute myeloid leukemia that induce complete remissions. Here, we investigate pre-clinical combinations with ML NK cells for MM, including blockade of the inhibitory NKG2A/HLA-E checkpoint as well as MM-specific targeting via myeloma-targeting monoclonal antibody (mAb) and chimeric antigen receptor (CAR) engineering. Human ML NK cells generated from healthy donors display IFN-γ responses similar to conventional (cNK) cells, and only modestly increased cytotoxicity against myeloma cell lines (MM1.s and OPM2). MM cell lines and primary MM cells express high levels of HLA-E, a major negative checkpoint for ML NK cells that binds to the inhibitory receptor NKG2A expressed on NK cells. Indeed, mass cytometry analysis of MM patients compared to healthy controls demonstrated enrichment of a NK cell population expressing high levels of NKG2A, but also high levels of activation and cytotoxic markers. Disruption of HLA-E binding to NKG2A with the blocking mAb monalizumab (AstraZeneca) significantly increased healthy donor ML NK cell IFN-γ response (P=0.016), degranulation (surface CD107a, P=0.017), and killing (P=0.009) of MM1.s when compared to isotype mAb. NK cells isolated from patients with MM showed similarly enhanced functionality after ML differentiation and NKG2A blockade (IFN-γ P=0.008). In an NSG mouse model of MM using MM1.s cells, healthy donor ML NK cells combined with monalizumab showed improved control of MM tumor growth compared to ML NK cells without monalizumab (P<0.001), conventional NK cells, or control tumors alone (P<0.001). To further improve recognition of MM, ML NK cells responses were assessed in combinationwith a mAb against SLAMF7 (elotuzumab) that both targets MM cells and activates NK cells. Elotuzumab treatment increased the functionality of ML NK cells against MM cell lines (IFN-γ, P=0.004) with an additive effect of both elotuzumab and monalizumab (IFN-γ, P=0.003). In an alternative strategy to enhance MM targeting, we engineered an anti-BCMA CAR (anti-BCMA/4-1bb/z) into ML NK cells. BCMA-CAR+ NK cells exhibited increased IFN-γ (P=0.001) production and degranulation (P<0.001) against MM1.s compared to non-transduced control NK cells. These functional responses were additionally enhanced with ML differentiation. Collectively, these findings suggest that ML NK cell differentiation, combined with strategies to address inhibitory checkpoints (NKG2A) or MM-specific activation (elotuzumab, anti-BMCA CAR), are promising cellular therapy strategies for MM patients. Citation Format: Alice Y. Zhou, Melissa M. Berrien-Elliott, Ravi Vij, Mark Fiala, Michelle Becker-Hapak, Lynne Marsala, Miriam T. Jacobs, Nancy D. Marin, Jennifer Tran, Jennifer Foltz, Pamela Wong, Julie Fortier, Sarah Kelley, Carly Neal, David Russler-Germain, Timothy Schappe, Todd A. Fehniger. Overcoming barriers to the natural killer cell response against multiple myeloma: Manipulating the NKG2A/HLA-E checkpoint and CAR engineering of memory-like natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2897.

Associations of granulocyte colony-stimulating factor with toxicities and efficacy of chimeric antigen receptor T-cell therapy in relapsed or refractory multiple myeloma

Rapid Response to Idecabtagene Vicleucel in a Myeloma Patient Refractory to Multiple Prior Lines of Anti-BCMA Directed Therapies