Hypoxia promotes BCMA loss and a suppressive secretome thereby hindering CAR T cell therapy in multiple myeloma

CAR T Cell Therapy Targeting G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), a Novel Target for the Immunotherapy of Multiple Myeloma

Targeting Cancer Associated Fibroblasts in the Bone Marrow Prevents Resistance to Chimeric Antigen Receptor T Cell Therapy in Multiple Myeloma

Targeting Cancer Associated Fibroblasts in the Bone Marrow Prevents Resistance to Chimeric Antigen Receptor T Cell Therapy in Multiple Myeloma

Trogocytosis May Attenuate the Efficacy of Anti-BCMA CAR T Cells Administered in Combination with γ-Secretase Inhibitor

Evolving Data on CAR T-Cell Therapy for Multiple Myeloma, Novel Targets, Conditioning Regimens and Novel Constructs: A Systematic Review

Purpose: Multiple myeloma is characterized by the expansion of malignant plasma cells in the bone marrow (BM), which is associated with excessive production of monoclonal immunoglobulins in blood and urine in patients. The addition of monoclonal antibodies as immunotherapies in MM has further improved patient outcome. However, MM remains incurable for most patients, since drug-resistant clones constantly emerge and evolve. BCMA has been found to be overexpressed in MM and is being explored as a target for both antibody based and live cell therapy. Method: BCMA expression in MM patients was quantified using RQ PCR, and soluble BCMA was quantified using ELISA. The consensus BCMA.ECD sequence was cloned into a vector, sequenced, and protein expression was carried out using the Expi293 system, characterized by SDS-PAGE. Mice were immunized, and hybridoma cells were generated for the production of Anti-BCMA.ECD antibodies, which were further purified and characterized using western-blotting and other assays. The best binders were identified, and the ScFv sequence was incorporated into a novel second-generation CAR (Chimeric Antigen Receptor) T-cell, which was screened using flow-cytometry. Results: We characterized BCMA expression in a cohort of MM patients and found it to be elevated at the mRNA level. Serum or soluble BCMA levels were also elevated, predicting inferior therapeutic outcomes in MM patients. BCMA was validated as a candidate for Anti-BCMA therapy. BCMA.ECD protein was expressed in a eukaryotic system and used to generate hybridomas, resulting in twenty different clones. These clones were screened for the best binders, characterized by their binding affinity to BCMA.ECD protein using ELISA. The best binder (9C4) was used for deciphering the ScFv sequence for the development of a novel second-generation CAR. Conclusion: This study led to the characterization of BCMA expression and variation in Indian MM patients and the development of an Anti-BCMA antibody for a second-generation CAR. Citation Format: Gunjan Dagar, Ashna Gupta, Ravi Chauhan, Kalpana Luthra, Mayank Singh. Design and Development of a Novel Anti-BCMA.ECD Antibody for Live Cell Therapy in Multiple Myeloma (MM) [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr P31.

In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.

Cardiac Glycosides Induce Immunogenic Changes in Myeloma Cells and Enhance the Efficacy of Monoclonal and Bispecific Antibody Therapy

Multiple myeloma (MM) is a malignant plasma cell disorder that remains incurable for most patients despite significant improvements achieved with modern therapy. Tumor evasion is a key process in the pathogenesis of MM and a compromised immune system is associated with more aggressive forms of the disease. In contrast, the emergence of myeloma-specific immune responses after both autologous and allogeneic stem cell transplantation is associated with better prognosis. Adoptive T cell therapies may improve specific anti-myeloma immunity resulting in long-lasting remissions. CAR T cell therapies for MM are at an early stage of clinical development. To date, anti-BCMA CAR T cells have shown the greatest results in early-phase clinical trials. Toxicities have included cytokine release syndrome (CRS) and neurotoxicity. Current areas of research in CAR T cell therapies include the use of gene-editing to enhance their effectiveness and safety, the integration of CAR T cells with other therapies (immunomodulatory drugs, checkpoint inhibitors) and CAR T cells to target multiple antigens.

Natural killer (NK) cells are specialized components of the innate immune system with an essential role in immune surveillance against multiple myeloma (MM). However, patients with MM often exhibit significant impairments in both the quantity and functionality of NK cells, particularly those who are refractory or have relapsed disease. Increasing evidence suggests that harnessing NK cells may provide a promising “off-the-shelf” therapeutic strategy for treating MM. Recent advancements in chimeric antigen receptor (CAR) engineering have enhanced the capabilities of NK cells, allowing them to target MM-associated antigens effectively. This innovative approach may help mitigate the off-target toxicities often associated with NK cells and address challenges such as the ability of MM cells to evade immune detection. CAR-NK therapy shows promise for B-cell malignancies, but its efficacy in MM has been less examined. This comprehensive review revealed that CAR-NK cells elicit a robust immune response against myeloma cells in preclinical studies conducted both in vitro and in vivo. Furthermore, the development of dual-CAR NK cell therapies indicates promise in overcoming myeloma escape mechanisms. Numerous clinical trials are currently underway to evaluate the efficacy and safety of CAR-NK cell therapy for individuals diagnosed with MM.

CD38BiTE-secretion overcomes antigen escape in BCMA-targeted CAR T cell therapy for multiple myeloma

NK cell therapy in relapsed refractory multiple myeloma

Characteristics of Clinical Trials for Multiple Myeloma and CAR-T Cell Therapy: A Cross Sectional Analysis

The promise of chimeric antigen receptor (CAR) T cell therapy in multiple myeloma

Multiple myeloma (MM) is one of the most frequent haematological malignancies characterized by production of paraprotein with constant isotype and light-chain restriction. Abnormal paraprotein bands (APBs) detectable by serum immunofixation electrophoresis (IFE) has been reported following autologous and allogeneic transplantation.1-4 Chimeric antigen receptor (CAR)-T cell therapy is an emerging and promising treatment for patients with relapsed or refractory (RR) MM with an overall response rate ranging from 73% to 98%.5, 6 Consistent with high-dose chemotherapy followed by transplantation, the lymphodepletion chemotherapy combined with CAR-T cell infusion also involves immune reconstitution. However, our understanding about the prevalence and clinical significance of APBs after CAR-T cell therapy is extremely limited. Until now, there is one available report of APB phenomenon that was observed in four patients with RRMM treated with anti-BCMA CAR-T cells.7 Here, we describe a case with RRMM who has experienced three different immunoglobulin (Ig) class switch after bispecific CAR-T cell therapy and maintained ongoing stringent complete response (sCR) for over 3 years without additional treatment. In January 2018, a 63-year-old female was diagnosed as MM, κ type, stage IIIa according to the Durie-Salmon staging criteria, stage II according to the revised international staging system, and intermediate risk according to the Mayo stratification (Data S1).8 The patient received four lines of chemotherapy, but her disease was not effectively controlled (Figure 1A). In August 2018, the patient enrolled the phase 1 clinical trial of bispecific BM38 CAR-T cell therapy in RRMM (Chictr.org.cn, ChiCTR1800018143).9 She received preconditioning chemotherapy followed by one infusion of BM38 CAR-T cells at the dose of 2 × 106 cells/kg. BM38 CAR-T cells peaked on day 14 after infusion and survived at a relatively low level at month 3 to 12 (Figure 1B). The patient achieved sCR on day 14 and maintained ongoing remission for over 3 years per 2016 IMWG criteria (Figure 1C; Table S1).10 The patient had pre-existing hypogammaglobulinemia, which continued to deteriorate after CAR-T treatment. To prevent infections, intravenous immunoglobulin at a dosage of 400 mg/kg every 2–4 weeks was administrated for 3 months post-infusion. The hypogammaglobulinemia improved at month 3 and returned to the normal levels at year 3 (Figure 1D). The patient experienced grade 4 hematologic toxicities that returned to the normal levels at month 3 (Figure 1E). Interestingly, we observed sequential changes of APBs detected by serum IFE, from κ chain at screening and baseline, to normal on day 14 and at month 2, then to κ and λ at month 3, next to IgM-λ at month 4, subsequently to IgG-κ and IgG-λ at month 5–9, and finally to normal at month 12 and maintained normal until the last follow-up (Figure 2A). To identify whether there were tumour cells below the detectable levels by flow analysis, we retrospectively analysed IgH rearrangements of sequential BM samples by next-generation sequencing and confirmed the absence of MM clones during Ig isotype switch after CAR-T infusion (Figure 2B; Tables S2–S5). It is important to clarify the clinical significance of APB occurrence for efficacy evaluation and patient management. The appearance of APBs in patients distinct from the original paraprotein could be attributed to either a change in paraprotein production by the malignant clone, or a temporary phenomenon of immune reconstitution. APBs occurred in the patient at month 3 following CAR-T infusion and lasted at month 9, consistent with previous study.7 At detection of APBs, the patient was asymptomatic and free of hypercalcemia, renal failure, anaemia, and bone pain. Simultaneous BM examination and IgH sequencing analysis excluded recurrence of malignant cells. Imaging examination excluded extramedullary lesions. These results suggested APBs observed in the patient were not associated with clinical relapse or malignant transformation. Therefore, we chose to carefully monitor the patient, rather than early administration of salvage therapy. Encouragingly, APBs disappeared at month 12, and the patient has attained sustained remission for over 3 years. Transient APBs are observed during the recovery of Ig production after transplantation, and in the setting APBs suggest a significant role for immunosurveillance in long-term suppression of myeloma clone.1-4 Nevertheless, B-cell reconstitution after transplantation recapitulates normal ontogeny but in a clonally dysregulated pattern, probably associated with an impaired T-cell regulation.11 ABPs occurrence coincided exactly with recovery of normal plasma cells in BM, serum Ig and peripheral blood cells. Hence, we supposed that the presence of APBs in the patient correlated with immune restoration. However, peripheral immune cell subsets were not assessed, which was one limitation of the study. Further studies are warranted to monitor immune cell subsets for better understanding the evolution of immune reconstitution in the presence or absence of APBs. The IgH switch from synthesis of IgM to IgG, IgA or IgE is mediated by class switch recombination (CSR).12 CSR occurs by intrachromosomal deletional recombination within switch regions, 2–10 kb DNA segments, located upstream of IgH constant region genes. Light chains are synthesized in an independent pathway.13 κ locus are located on chromosome 2 and λ locus on chromosome 22, different from heavy chain genes, which are located on chromosome 14. λ locus rearrangement occurs only when the rearrangements of both κ locus are ineffective. Two possible mechanisms are raised based on these findings: one is “intraclonal class switch” hypothesis that some unknown molecular genetic events promote Ig class switch, and the other is “different progenitor B cell lines” hypothesis that transient imbalance of B and Ig-producing plasma cells under pressure of BM38 CAR-T cells at month 3 to 12 leads to temporary occurrence of APBs.14, 15 In summary, we report a relatively rare case of RRMM who experienced multiple Ig isotype switch after bispecific CAR-T cell therapy with an incidence of ABPs of 4.3% in our trial (n = 23).9 Our case suggests that the APBs are associated with immune restoration rather than malignant transformation. In this setting, APBs occurrence indicates a better outcome of MM patients based on our case and previously studies.1-4, 7 Careful monitoring is recommended to distinguish APBs caused by immune reconstitution or malignant recurrence. Further studies need to be carried out to illustrate their pathogenesis and mechanisms. Heng Mei and Yu Hu designed and supervised the study; Heng Mei and Chenggong Li enrolled patients and took care of the patients; Lei Chen and Jun Deng contributed to laboratory tests; Chenggong Li and Yun Kang collected clinical data; Chenggong Li, Jiachen Liu, Heng Mei, and Wenjing Luo analysed data, wrote and revised the manuscript; Chenggong Li and Jia Xu contributed to figures proofreading. The authors thank Zhejiang Cellyan Biotechnology Co. Ltd for BM38 CAR-T cell production; the patient and her families, friends, and caregivers and all the study staff and health care providers on coordination; all the faculty and staff at the Institute of Haematology, Union Hospital, Huazhong University of Science and Technology, for patient care and laboratory tests. This work was supported by grants to Heng Mei from the National Key R&D Program of China (No. 2019YFC1316204) and the National Natural Science Foundation of China (No. 81873434, No. 82070124) and Natural Science Foundation of Hubei Province (No. 2020CFA065). The authors declare no competing financial interests. This study was registered on Chictr.org.cn, number ChiCTR1800018143. The patient provided written informed consent. Table S1 Table S2 Table S3 Table S4 Table S5 Data S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.