Multiple Myeloma Therapy Research Articles

Unraveling the Dynamics of T Cell-Dependent Bispecific Antibodies in Multiple Myeloma: Multi-Omic Insights from Primary Patient Cells

Introduction: Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Despite significant advancements in immuno-oncology (IO) therapeutics, such as T-cell dependent bispecific (TDB) antibodies targeting MM antigens, MM remains incurable. This highlights the need for further research to develop personalized and effective treatments. The study of primary MM cells is pivotal for understanding therapeutic effects and patient variability but faces challenges due to cell scarcity and low ex vivo viability. Our microfluidic platform, requiring minimal cell input and no matrices or immobilization, maintains viability and offers versatile, integrated readouts, thereby deepening the breadth and depth of information from ex vivo experiments. In this study, we developed a multi-omic assay to gain insights into cytotoxicity, cytokine profiling, and gene expression changes to conduct a comprehensive analysis of TDBs targeting MM antigens in primary MM co-cultures. Methods: Primary T cells and MM cells, isolated from cryopreserved patient samples, were cultured in the microfluidic platform, either autologously or allogeneically, at specific effector-to-target cell ratios. Cells were treated with TDBs with daily media replenishment for up to 72 hours. Following incubation, cells were stained with Hoechst, viability staining, CD25, and CD69 antibodies followed by high-content imaging. Supernatant from each condition was pooled for cytokine profiling, and nucleic acids were extracted from the cells post-imaging. Gene expression analysis was performed using NanoString's PanCancer IO 360™ panel. Results: Image analysis revealed that TDB treatment induced T cell-mediated cytotoxicity in four patients, with sub-optimal TDB-induced cytotoxicity observed in two patients, highlighting intrinsic resistance mechanisms to TDB treatment and the need for greater mechanistic understanding. Notably, all patients' effector cells exhibited upregulation of CD69, a T cell activation marker, after 24 hours post-TDB treatment which was sustained for 72 hours. TDB treatment also led to the upregulation of CD25, starting at 24 hours and peaking 72 hours post-treatment. Furthermore, enhanced secretion of interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 (IL-2) was detected in samples that responded to TDB treatment, while samples that exhibited resistance to TDB treatment showed no IL-2 secretion. Gene expression analysis revealed an upregulation of genes linked to the interferon-gamma signaling pathway in TDB-treated samples. Additionally, when comparing the gene signatures between responders and non-responders at baseline, the non-responders' samples displayed increased expression of CD3D, CD3G, CD8a, and CD28. These markers, typically associated with T-cell signaling, hint at a more complex pre-existing T-cell activation landscape in non-responders, potentially influencing the outcome of T-cell engager therapies. Discussion: We introduced a novel assay on our microfluidic platform to profile TDBs with primary MM and immune cells. This platform offers several key advantages. First, it enables robust dose-response evaluations of cytotoxicity, providing accurate assessments of the TDBs' effectiveness. Second, it allows for concurrent and longitudinal profiling of cytokine release and measurement of T cell activation markers, offering a comprehensive understanding of T cell responses. And third, the platform facilitates gene expression analysis using the same cells, enabling the generation of fully integrated readouts and deeper mechanistic understanding. This multi-omics approach not only deepens our understanding of IO therapies for MM but also offers potential for diverse hematological malignancies. Direct analysis of patient cells provides invaluable insights into individual responses, setting the stage for mechanistic discoveries and the development of more effective, personalized therapies. Ultimately, these insights underscore the transformative potential of integrative multi-omics in steering immune-centric therapies for hematologic malignancies.

Cells of the Osteoblast Lineage Cross-Talk with Myeloid-Derived Suppressor Cells to Promote Multiple Myeloma Growth

Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM) and is highly influenced by the BM tumor microenvironment. Osteolytic bone disease is a hallmark of MM and is partly due to osteoblast (OB) inhibition. MM cells suppress osteoblast maturation and function via inhibition of the critical Runt-related transcription factor 2 (Runx2) in immature OBs. However, immature cells of the osteoblast lineage (e.g. pre-osteoblasts) remain present and even increased in the MM bone microenvironment and their contribution to the progression of MM has not been well understood. We previously developed a syngeneic mouse model of MM in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2 -/- mice), and demonstrated that OB-Runx2 deficiency creates a highly inflammatory BM microenvironment that is responsible for MM cell bone-homing to new bone sites. We therefore hypothesize that pre-osteoblasts play a role as promoters of MM cell survival and proliferation. To determine the direct effect of pre-osteoblasts on MM cells, we established co-cultures of pre-osteoblasts with MM cells. 5TGM1 murine MM cells were cocultured with MC3T3 murine pre-osteoblasts and newborn mouse calvarial pre-osteoblasts respectively. Within 48hrs of co-culture, each of pre-osteoblast populations slightly stimulated proliferation of 5TGM1 MM cells. These data suggest that pre-osteoblasts do not directly affect myeloma growth in vitro. Therefore, we next focus our studies on the role of pre-osteoblasts in MM progression in vivo. Firstly, we used OB-Runx2 deficient mouse model to assess the impact of pre-osteoblast accumulation on MM progression in vivo. 2x10 5 5TGM1-Luciferase MM cells were injected into the right tibia of OB-Runx2 -/- mice and control mice. Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1 MM cells) ELISA demonstrated that, compared with controls, OB-Runx2 -/- mice had a much larger tumor burden. Following, to more thoroughly evaluate the role of pre-osteoblasts in early stage of MM cell engraftment in bones, we used MC3T3 murine pre-osteoblasts to modulate pre-osteoblast populations at the sites of MM engraftment. 2x10 5 5TGM1-Luciferase MM cells were injected directly into the right tibia of NSG mice with or without equal numbers of MC3T3 pre-osteoblasts. Tumors grew significantly larger when co-injected with 5TGM1 MM cells and MC3T3 pre-osteoblasts than injected with 5TGM1 MM cells alone, which was confirmed by luciferin imaging and serum IgG2bκ ELISA. These data suggest that pre-osteoblasts provide critical support for MM progression in vivo. Given that pre-osteoblasts have no direct effect on the growth of MM cells in vitro, we hypothesized that the pre-osteoblastic niche may regulate MM cells via other intermediary cells in BM. We previously showed that OB-Runx2 deficiency induces an immunosuppressive BM microenvironment that is marked by an increase of myeloid-derived suppressor cells (MDSCs). To investigate whether pre-osteoblasts support MM cell survival and proliferation through the functional crosstalk with MDSCs, 5TGM1 MM cells were co-cultured with MC3T3 pre-osteoblasts and mouse calvarial pre-osteoblasts respectively in the presence or absence of MDSCs. Interestingly, pre-osteoblasts significantly promoted the proliferation of MM cells under the existence of MDSCs. To extend these findings in vivo, we next determined whether MDSC depletion can alleviate the promoting effect of pre-osteoblasts on MM progression. Gemcitabine (GEM) is an FDA-approved anti-cancer agent and MDSC inhibitor used in the treatment of solid tumors but not commonly used in MM therapy. We again injected 5TGM1-Luciferase MM cells into the right tibia of NSG mice with or without co-injection of MC3T3 pre-osteoblasts. We treated tumor-bearing mice with PBS or GEM (i.p. 30 mg/kg/week) for 4 weeks. Bioluminescence imaging and serum IgG2bκ ELISA demonstrated that GEM could overcome the proliferative effect of pre-osteoblasts on MM cells in mouse model, and this effect was independent of NK, B and T cells. Taken together, using in vivo and in vitro models, we show that pre-osteoblasts have a positive regulatory impact on MM cells through communication with MDSCs. Ongoing mechanistic studies aim to delineate how pre-osteoblasts modulate the functions of MDSCs to promote MM progression.

Small Molecule Inhibition of MAGE-A3 Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma

Background: The Type I Melanoma Antigen Gene-A3 (MAGE-A3) is commonly expressed in multiple myeloma (MM), a cancer of plasma cells. MAGE-A3 binds to Kap1 to form ubiquitin (Ub) ligase complexes and is associated with inhibition of apoptosis, resistance to chemotherapy, and cell cycle dysregulation. The highly conserved MAGE Homology Domain (MHD) undergoes a conformation change from “closed” to “open” in order to complex with Kap1, activate Ub ligase activity, and bind substrates. RNA interference (RNAi) knockdown of MAGE-A3 in MM cells results in upregulation of the endogenous CDK4/6 inhibitor P21 Cip1 and pro-apoptotic BIM, resulting in cell cycle arrest and intrinsic apoptosis. Knockdown of MAGE-A3 also increased cell death in response to melphalan and panobinostat, but not to bortezomib or cis-platin. These findings lead to the hypothesis that small molecule inhibition (SMI) of MAGE-A3 Ub ligase activity will have therapeutic application in this disease. Methods: We used the Schrodinger suite to perform in silico screening of a custom designed library of compounds using the crystal structure of the MAGE-A3 MHD to identify potential binding molecules that stabilize the closed MHD conformation. Candidates were then screened on human myeloma cell lines (HMCL) by CellTiter-Glo assay to identify compounds that reduced cell viability. Isothermal titration calorimetry was used to identify candidate SMI that bound to the MHD. HMCL were co-incubated with SMI; cell cycling was assessed by BrdU assay and apoptosis by Annexin V staining. Protein expression of p21 Cip1, BIM, and MAGE-A3 was assessed by Western blot. Combinatorial indexes in co-incubation experiments with ergotamine and chemotherapy agents were calculated with the Chou-Talalay method using CompuSyn. Results: Structure-based and biochemical screening nominated ergotamine as a candidate MAGE-A3 SMI. Ergotamine bound to the MAGE-A3 MHD and induced cell cycle arrest and apoptosis in MAGE-A3-expressing human myeloma and lymphoma cell lines and increased expression of pro-apoptotic BIM and p21 Cip1, similar to results with RNAi knockdown. These effects were not observed in negative control MAGE-A3(-) lymphoma cells. Co-incubation of ergotamine with chemotherapy agents melphalan or panobinostat resulted in synergistic killing of MAGE-A3+ HMCL. Ergotamine did not synergize with bortezomib or cis-platin under these conditions. Conclusions: These results support the combined structural and biochemical methods as an efficient means of screening for candidate inhibitors for MAGE-A3. Ergotamine recapitulated the results of RNAi knockdown of MAGE-A3 in cells that express the target, supporting a tool compound for further pharmacologic development. MAGE-A3 SMI have the potential to be a novel class of MM therapy that may be effective alone or in combination with chemotherapy or immune oncology agents. MAGE inhibitors may have broader application in other target-expressing cancers, including lung, melanoma, breast, and ovarian cancers, which are among the most common causes of cancer death.

Ixazomib Maintenance Therapy in Chinese Patients with Newly Diagnosed Multiple Myeloma Not Treated with Stem Cell Transplantation: A Single-Arm, Open-Label Study

Introduction: Multiple myeloma (MM) is a life-threatening cancer of plasma cells in the bone marrow. It often leads to bone marrow failure and bone destruction. Lenalidomide is the only drug approved by Food and Drug Administration (FDA) and European Medical Association (EMA) as maintenance therapy for MM post stem cell transplantation (SCT), however it is not approved as maintenance for patients who are ineligible for high-dose therapy followed by SCT and is also not suitable for all patients due to toxicity and new primary malignancy (NPM) concerns. Hence, new maintenance agents are needed to offer patients continuous disease control while maintaining their health-related quality of life (HRQoL). Ixazomib, an oral protease inhibitor, was approved by FDA for MM patients with at least one prior therapy. The present study assessed the long-term safety, tolerability and efficacy of ixazomib maintenance therapy in Chinese newly diagnosed MM (NDMM) patients who had not undergone SCT. Methods: The C16021 CCS (NCT03748953) was a single arm, open-label extension of the TOURMALINE-MM4 (NCT02312258) phase 3 clinical trial, and was conducted from June 2018 to October 2022. It included Chinese patients aged ≥18 years with a confirmed diagnosis of symptomatic NDMM who had achieved a major response [complete response (CR), very good partial response (VGPR), or partial response (PR)] with initial MM therapy. Patients received oral ixazomib capsules at days 1, 8, and 15 of every 28-day cycle. The dose of ixazomib was escalated from 3 mg to 4 mg if it was tolerated during the first 4 cycles. The duration of treatment was approximately 24 months or until patients experienced progressive disease (PD) or unacceptable toxicity, whichever occurred first. The primary objective was to assess long-term safety and tolerability of ixazomib. The secondary endpoints included progression free survival (PFS), overall survival (OS), time to progression (TTP), time to next line therapy (TTNT), incidence of new primary malignancy (NPM) and HRQoL. Results: A total of 37 patients were included in the study from 7 study centers in China. Of these 37 patients, 35 patients were included in the safety analysis. The dose was escalated to 4 mg at day 1 of cycle 5 in 77% of patients. A total of 34 patients (97%) experienced at least 1 treatment emergent adverse events (TEAE) with 30 patients (86%) experiencing a treatment-related TEAE and 16 patients (46%) experienced a Grade ≥3 TEAE. The most common Grade ≥3 TEAEs reported were thrombocytopenia (2 patients, 6%) and pneumonia (2 patients, 6%). No NPMs were reported. SAEs were reported in 7 patients (20%) and 2 patients (6%) had their study drug discontinued permanently due to a TEAE. Three patients (9%) experienced TEAEs of peripheral neuropathy (PN, all grade 1 or 2) and all 3 patients had resolution and improvement of PN events during the treatment period. The median time to resolution of PN events was 24.0 days and median time to improvement of PN events was 8.0 days. Overall, 33 patients were included in the efficacy analysis. The median age was 68.3 years, male 64% and female 36%, ISS stage at initial diagnosis was 21%, 30% and 48% for stage I, II and III, respectively. The median time from initiation of induction therapy to first dose of study drug was 8.9 months. With a median follow-up of 15.9 months, median PFS and TTNT were 21.3 months and 24.7 months, respectively. If counting from induction therapy, median PFS was 30.0 months. All patients were still alive at date of last contact. Three patients (9%) demonstrated deepening of response during treatment. Of note, since only 9 patients (27%) had VGPR or PR at study entry, the potential for assessing deepening of response in this study was limited to these 9 patients. Ixazomib maintenance treatment showed no negative impact on HRQoL, as measured by the EORTC QLQ-C30, EORTC QLQ-MY20, EQ-5D-5L, and EQ VAS. Conclusion: Ixazomib maintenance therapy showed a tolerable safety profile with prolonged PFS in Chinese patients and the response was maintained thus providing a valuable treatment option in NDMM patients who have not undergone SCT. The results of this study were consistent with TOURMALINE-MM4 global study.

Suppression of TRIP13 Induces Metabolic Changes and Potentiates Ferroptosis in Multiple Myeloma

Introduction Despite the remarkable success of combination treatments, multiple myeloma (MM) still poses significant challenges, with a high percentage of patients experiencing relapse over time. Additional metabolic alterations are found in relapsed and refractory MM; however, their impacts have not been fully elucidated. Expression of thyroid hormone receptor interactor 13 (TRIP13) has been identified by Gene Expression Profile (GEP)-70 to correlate with poor prognosis in MM. TRIP13 is an AAA ATPase that regulates the spindle assembly checkpoint in mitosis. Based on GEP data from 34 paired baseline and relapse samples, TRIP13 continuously increased from baseline to first and to second relapse. Overexpression of TRIP13 induces bortezomib resistance. In this study, we aimed to investigate how TRIP13 might regulate MM cell metabolism and to use this information to develop a novel combination strategy to overcome MM resistance. Methods Knockout (KO) of TRIP13 in OPM2 and MM1.S cells was generated by dual gRNAs CRISPR-Cas9 editing deleting a fragment between exon 4 and exon 5. Cell growth curves and response to bortezomib were recorded in the control and TRIP13-KO cells. Metabolomics was performed to reveal cellular metabolic alterations. Seahorse analysis was used to test the mitochondrial stress and glycolysis stress. To investigate whether TRIP13 KO promotes ferroptosis, an intracellular iron-dependent cell death characterized by the accumulation of lipid peroxides, we applied C11-BODIY staining after treatment with Erastin, a ferroptosis-inducer. Expression levels of cyclooxygenase 2 (encoded by PTGS2, a hallmark of ferroptosis), cystine transporter (xCT), and intracellular glutathione peroxidase 4 (GPX4) were analyzed. In addition, the protein levels of iron metabolic molecules, including iron importer transferrin receptor protein 1 (TFRC), iron exporter ferroportin 1 (FPN1), ferritin heavy chain 1 (FTH1), and nuclear receptor coactivator 4 (NCOA4) were assessed. To study whether TRIP13 KO delays MM progression in an immune-competent MM mouse model, Vκ12653 murine MM cells were genetically modified by CRISPR-mediated TRIP13-KO and then injected into C57BL/6 mice. Mouse survival was monitored. Results The cell-doubling rate of TRIP13-KO cells was decreased by 50% compared to control cells. After 24 hours of treatment with 5 nM bortezomib, cell viability of TRIP13-KO cells decreased by 31% compared with control cells. The metabolomic studies revealed that TRIP13-KO OPM2 and MM1.S cells both had significantly decreased intracellular L-serine (FC = 0.57) but increased pyruvate (FC = 1.79), citric acid (FC = 1.76), and isocitric acid (FC = 1.64), indicating TRIP13 may regulate serine metabolism, mitochondrial function, and cellular redox balance. Seahorse analysis indicated that TRIP13-KO MM cells showed decreased glycolysis (decreased by 30%, p < 0.001) and glycolytic capacity (decreased by 33%, p < 0.001) while oxidative phosphorylation showed little change. Moreover, lipid peroxidation and PTGS2 were increased in TRIP13-KO cells after Erastin treatment, indicating that these cells were more vulnerable to ferroptosis. Mechanistically, inhibition of TRIP13 decreased GPX4, FPN1, FTH1, and increased NCOA4, while overexpression of TRIP13 showed the opposite changes, suggesting that targeting TRIP13 sensitizes MM cells to ferroptosis by impairing cellular redox balance and increasing the labile-iron pool. Lastly, TRIP13-KO significantly prolonged mouse survival in the Vκ12653 mouse model (median survival: TRIP13-KO 93 days vs. Control 60.5 days, p < 0.001). Conclusion Our study revealed that TRIP13 plays a critical role in regulating MM cell metabolism. By suppressing TRIP13, we observed increased sensitivity of MM cells to bortezomib, leading to enhanced ferroptosis-mediated cell death and prolonged mouse survival. These findings strongly suggest that targeting TRIP13 could be a promising approach in MM therapy. Moving forward, our future investigations will concentrate on unraveling the precise molecular mechanisms underlying TRIP13-mediated alterations in cell metabolism. Additionally, we plan to explore the potential synergistic effects of TRIP13 inhibition in combination with metabolic targeted treatments, such as venetoclax, to further enhance therapeutic outcomes in MM.

Examining Racial Disparities in Multiple Myeloma Research and Treatment

Multiple myeloma (MM) is a disease which occurs in all races, geographic locations, and ethnicities, however the burden of this cancer is especially prominent in the African American/Black population with prevalence estimated to be as high as two to three times that of the White population. As a result MM is the most common hematological malignancy in Black patients. Despite a higher disease burden, this population suffers disproportionate rates of mortality. From 2017-2022 Black men had a 6% higher incidence rate than White men, but a 19% higher mortality rate. This disparity in outcomes between ethnic groups is likely multifactorial. Some explanations include novel therapy underutilization, or prevalence of comorbid conditions. It may also be attributable to biological differences, such as an increase rate of immunoglobin A disease, and an increased rate of chromosome 14 translocations. There may as a result be a heterogenous response to novel MM therapy. Unfortunately, clinical trial participation of Black patients has been significantly underrepresented. Studies that sought to analyze the results of novel therapy trials in this population found a trend towards decreased efficacy, however, were unable to reach significance due to insufficient study participation. We conducted a pooled analysis of interventional phase III clinical trials obtained from ClinicalTrials.gov data from 01/01/2000 to 12/31/2022. Studies who did not include racial/ethnicity data were excluded. There were 34 trials included in total that met this criteria and included in this analysis. There were 16,405 total enrolled patients analyzed. 13,618 (83.01%) were White, 1,255 (9.21%) were Asian, 777 (4.74%) were Black/African American, and 454 (2.7%) identified as Hispanic, with the remaining identified as other. Study participation was analyzed over time. Percentage of patients enrolled by year showed a negative correlation in Black/African Americans over the analyzed period. Our analysis suggests greater efforts need to be made to ensure the enrollment of a more diverse and representative MM population. Although this disease is roughly twice as prevalent in Black populations, clinical trial study participation since 2003 involved 18-times more White than Black patients. Despite ongoing recognition of this problem, study participation in MM has continued to remain largely homogenous. Interventions that may improve this problem include fostering and rebuilding trust in the clinical trial system that was damaged from historical abuses, expanding clinical sites to more diverse areas, expanding/revising eligibility criteria, and employing changes to enrollment techniques that better represent the demographics of the population intended to treat. Ultimately, the clinical trials for an intervention should appropriately reflect the population who will use it, otherwise these novel treatments may have unexpected heterogeneity in efficacy and safety once employed clinically.

Development of a Novel, Allogeneic GPRC5D-Directed CAR for Treatment of Multiple Myeloma Patients

Despite the success of BCMA-targeted therapy in advanced multiple myeloma, the disease is still considered incurable. Relapse following BCMA-directed cellular and antibody-based therapies is commonly associated with decreased surface expression of BCMA on malignant plasma cells. The G protein-coupled receptor, class C group 5 member D (GPRC5D) represents an additional target whose expression is limited to bone marrow and hair follicles, with selectively increased expression on malignant plasma cells. Thus far, the use of GPRC5D-targeted CAR T cells has provided a strong clinical benefit in multiple myeloma with limited on-target, off-tumor effects. To this end, we have validated novel, fully human GPRC5D-specific CARs for use with our hypoimmune (HIP), allogeneic CAR T cell platform. This will allow rapid delivery of allogeneic GPRC5D CAR T cells from healthy donors that simultaneously circumvent “non-self” recognition through TCR, B2M and CIITA gene disruption and promote persistence through innate immune evasion by leveraging CD47 overexpression. For early selection of candidate GPRC5D binders, T cells from healthy donors were transduced with VSV-G pseudotyped lentivirus containing second-generation, fully-human GPRC5D CARs. A clinically validated GPRC5D CAR was produced in similar fashion and used as benchmark control for initial characterization experiments. Transduction efficiency of GPRC5D CARs was analyzed via flow cytometry. GPRC5D CAR efficacy was analyzed in vitro via 24-hour luciferase or long-term Incucyte cytotoxicity assays against RPMI-8226 and MM.1S multiple myeloma target cells. Systemic MM.1S in vivo tumor models were performed in NSG mice to further assess efficacy of lead CAR candidates. Here we show our candidate GPRC5D CARs demonstrate in vitro cytotoxicity and effector cytokine production that is comparable to clinically validated benchmark controls. In 5-day Incucyte cytotoxicity assays, GPRC5D CAR T cells expand up to 10-fold and reduce tumor cell counts at starting E:T ratios of 1:8. Furthermore, in MM.1S in vivo tumor challenge models, these GPRC5D CARs significantly reduce flux and prolonged survival relative to tumor only controls with tumor clearance that was in line with clinical benchmark controls. Using ex vivo VSV-G production, our GPRC5D CAR T cells control multiple myeloma tumor cells both in vitro and in vivo, demonstrating efficacy that is on par with GPRC5D clinical benchmark CAR T cells. Studies to further define the efficacy of HIP-modified GPRC5D CAR T cells are ongoing. Moving forward, further interrogating GPRC5D CAR activity against multiple myeloma tumor cells with varying levels of target antigen expression will be critical to understanding the benefit of deploying GPRC5D CAR T cells within the multiple myeloma clinical landscape.

BCMA Re-Emergence As a Prognostic Indicator of Clinical Relapse after Chimeric Antigen Receptor T-Cell Therapy (CART) in Multiple Myeloma

Background: The outcomes of patients with triple and penta-class relapsed refractory multiple myeloma (RRMM) are poor. B-cell maturation antigen (BCMA)-directed chimeric antigen receptor T-Cell (CAR-T) therapies in RRMM have been shown to improve outcomes since approval in 2021; however, relapses are common. We previously reported that patients lost BCMA expression after BCMA-directed therapy. Early re-emergence of surface BCMA on plasma cells (PC) in bone marrow (BM) may be a prognostic indicator for clinical relapse. Furthermore, there is limited published literature about clinical factors that increase risk of early BCMA re-emergence as well as clinical relapse post-CAR-T therapy. Methods: The study is a prospective cohort study that includes RRMM patients who underwent BCMA CAR-T at the University of Kansas Health System between May 2021 and May 2023 and were followed at regular intervals for the first-year post-infusion. All patients had standard procedure of BM evaluation at 1, 3, and 6 months, and 1 year. Eight-color MFC was performed on the BM specimens. BCMA antibody was purchased from R & D Systems (Minneapolis, MN). Data analysis was performed using FCS Express 5 software (De Novo Software, Los Angeles, CA). 500,000 to up to 2,000,000 events were acquired in all the cases. Responses were evaluated using the International Myeloma Working Group criteria. BCMA re-emergence was defined as BCMA greater than nadir on either polyclonal or monoclonal BM plasma cells. Results: 57 patients were evaluated and 44 patients (77 %) who lost their PC BCMA expression by multicolor flow cytometry (MFC) on bone marrow biopsy (BMBX) at Day 30 from baseline were included in the analysis. Demographic data of all 44 RRMM BCMA CAR-T recipients is shown Table 1. Median follow-up was 7 (4-14) months. Of the 44 patients, BCMA re-emergence was as follows: at 3-months (n=14, 32% of total sample), 6-months (n=5, 11%), 9-months (n=0, 0% of total sample), and 12-months (n=5, 11% of total sample). 14 (32%) of the 44 total patients had clinical relapse within 12 months. Of the 14 patients, 11 (79%) had BCMA re-emergence at any time point. 10 out of 11 (91%) of these patients had BCMA re-emergence before or at the same time as clinical relapse. The median time to BCMA re-emergence was 3 months and the median time to clinical relapse was 6 months. Only 1 patient had clinical relapse prior to BCMA re-emergence.Furthermore, at the 6-month mark, there were no significant clinical or demographic factors between those who relapsed vs. those who had a response to CAR-T therapy other than BCMA re-emergence (p-value 0.0445, Table 1). Conclusion: BCMA re-emergence on BM detected by MFC may be a prognostic indicator for clinical relapse in RRMM recipients of BCMA CAR-T therapies. It may be valuable to carry out close monitoring that includes evaluation of BCMA expression on BMBx at regular intervals post CAR-T therapy. Additional prospective studies with larger cohorts are needed to determine the risk factors for early BCMA re-emergence and clinical relapse in RRMM patients who receive BCMA CAR-T therapy.

Hyperbaric Oxygen (HBO) Effects on Blood Count Recovery and Post-Transplant Outcomes Following High-Dose Therapy and Autologous HSPC Transplantation for Multiple Myeloma: A Multicenter Phase II Randomized Clinical Trial

Background: By reducing systemic erythropoietin (EPO) levels, in vivo studies showed that hyperbaric oxygen (HBO) promoted homing of transplanted umbilical cord hematopoietic stem/progenitor cells (HSPC) to the bone marrow. In a pilot study, we demonstrated that HBO in autologous HSPC transplantation (auto-HSPC) was well tolerated. Time to absolute neutrophil (ANC) recovery was correlated with HBO-mediated reduction in EPO levels. Methods: In this phase II multicenter clinical trial, patients with multiple myeloma (MM) receiving high-dose melphalan and auto-HSPC were randomized 1:1 between HBO and no HBO. HBO was given as 100% oxygen, 2.5 ATA for a total of 90 min, in a single treatment on Day 0, 6 hours before cell infusion. The study's primary objective was to evaluate the effect of HBO on blood count recovery, filgrastim use, blood transfusions, and length of hospitalization (LOH). Exploratory objectives include HBO effects on EPO and IL-15 measured at several time points (pre-conditioning, Day 0 pre-HBO, 6- and 8-hours following HBO, and Days 1-3, 7 and 15 post-transplant). In addition, we explore HBO effects on Day 15 NK cell recovery. Results: A total of 99 patients were enrolled, with 52 randomized to HBO. Baseline characteristics were well balanced except for cell dose, with higher odds that patients with lower cell dose were treated with HBO. There was insufficient evidence of a difference between the two groups regarding time to neutrophil recovery, filgrastim use, blood transfusions, or LOH. Patients randomized to receive HBO were 1.7 (95% CI: 1.1-2.7) times more likely to have lymphocyte recovery (ALC) to > 0.5 k/µL by any given day compared to control (Figure-1). EPO levels (only University of Rochester Medical Center patients) were lower for HBO versus placebo on Day 1 (unadjusted p = 0.02), but there was insufficient evidence of a difference at other time points (Figure-2). Regarding immune reconstitution, IL-15 levels were not significantly different between the two groups. Significant correlation was found between Day 2 IL-15 levels and ALC recovery (p=0.01). We observed higher median NK cell count at Day 15 in the HBO arm (246 vs. 183), but this did not reach statistical significance (P=0.59). Conclusions: HBO resulted in lower EPO levels at Day +1 of auto-HSPC, translating to faster ALC recovery with no effect on neutrophil recovery. Longer follow-up is needed to determine if this will impact progression free survival. Higher NK cell recovery in the HBO arm compared to the non-HBO was not significant to account for the improvement in ALC recovery in the HBO arm. A pilot study (NCT04862676) is ongoing to determine if multiple HBO treatments can keep EPO levels low beyond Day +1 and impact outcomes.

Upfront Radiation Therapy (RT) for Post-Stem Cell Transplant Multiple Myeloma (MM) Patients with Oligo-Relapse/Progressive Disease: Factors Associated with Favorable Outcomes

Introduction: Relapsed or refractory disease post-stem cell transplant (SCT) for Multiple Myeloma (MM) can be clinically challenging. For localized relapses, radiation therapy (RT) can represent an attractive treatment modality. However, data on outcomes with RT remains scarce. We investigated the impact of RT as a treatment modality in the setting of oligo-relapse and oligo-progressive plasmacytomas after SCT and explored prognostic factors associated with favorable long-term outcome Methods: In this single-institution retrospective analysis, we identified 31 MM patients who underwent upfront RT for oligo-relapse or oligo-progressive MM after SCT between 2000 and 2021. Patients were excluded if they had received systemic therapy for evidence of concurrent biochemical relapse prior to RT start. The treatment responses to stem cell transplantation (SCT) and RT were assessed following the response criteria established by the International Myeloma Working Group (IMWG), which includes the categories: complete response (CR), very good partial response (VGPR), partial response (PR), minimal response (MR), stable disease (SD) and progressive disease (PD). Additionally, we utilized the R-ISS staging system to classify patients into Standard-Risk (R-ISS I), Intermediate-Risk (R-ISS II), and High-Risk (R-ISS III) groups, to allow for a comprehensive evaluation of treatment efficacy and outcomes. Results: Disease and treatment characteristics for the cohort are summarized inFigure 1. Of the 27 patients with imaging available prior to SCT, 67% (18) were assessed with PET, 22% (6) with skeletal survey, 3.7% (1) with MRI/CT, 3.7% with CT, and 3.7% (1) with x-ray. Following SCT, 48% (13) of patients were noted to have radiographic progression, 23% (7) had sCR, 23% (7) had a CR, 10% had a serologic CR, and 45% (14) had a VGPR. Among the 18 patients with radiographic evaluation (in addition to bone marrow biopsy and/or labs) post-SCT, 67% (12) were in radiographic CR at time of re-staging. Median times from SCT to new site of oligo-relapsed disease (20, 64.5%) and pre-existing but inactive sites of oligo-relapsed MM (4, 12.9%) were 31 months (3.2-117.5) and 9 months (3.4-15.9), respectively. Oligo-progressive MM, defined as active sites of disease both pre- and post-SCT were present in 22.6% (7) of patients. Prior to RT, 94% (29) had PET/CT imaging, with a median SUV of 4.4 (2.6-7.2) for target lesions. Solitary bone (77%) and extramedullary (23%) disease were treated to median dose of 30 Gy (8-49 Gy). At first disease response assessment post-RT, 77% (24) of which was done with PET/CT imaging , 42% (13) achieved CR. Among the 18 patients that did not achieve CR, 15 subsequently achieved CR at a median time of 7 months (1.7-44.5). A majority of patients (87%) received systemic therapy post-RT, with a median number of lines of therapy post-RT of 3 (1-17). The remaining 13% (4) of patients did not receive additional systemic therapy post-RT and did not experience progression. Median overall survival for the full cohort was 38 months (3.5-37.9). Conclusions: Utilizing modern systemic therapies in combination with post-transplant RT for patients with limited refractory or relapsed MM is a promising therapeutic approach. For those that remain refractory or relapse in a single site RT local control merits consideration supported by our report. Further investigation to identify patients at highest risk of progression and who may therefore benefit most from RT would be valuable. We plan to conduct additional analyses on the radiographic characteristics that may portend high risk disease, with the ultimate goal of developing well-defined criteria for giving RT earlier.

Therapy Related Multiple Myeloma: A Distinct Entity

Background The development of therapy-related multiple myeloma (t-MM) is a poorly described entity. Multiple myeloma (MM) is the second most common hematologic malignancy. Despite significant strides in MM treatment over recent decades, identifying modifiable risk factors, optimizing screening methodologies, and identifying potential treatment strategies or targets to address t-MM remains of utmost importance, as this disease continues to pose a significant therapeutic challenge and remains incurable. This retrospective review seeks to explore the incidence and characteristics of t-MM at a medium-sized, academic center. Methods We performed a single institution retrospective chart review of patients diagnosed with multiple myeloma between 2008-2023 and evaluated for prior diagnosis of solid malignancy, excluding all skin cancers, lymphomas or myeloproliferative neoplasm (MPN). We collected data for the following: prior treatment for solid malignancy, lymphoma or MPN, lapsed time between prior therapy and development of MM, cytogenetics, prominent subtype present, renal failure and hypercalcemia if present at time of diagnosis, management of t-MM including transplant data, disease progression including relapse status, and survival data. Results In our study, we identified a subgroup of 17 individuals who had a previous history of solid malignancy, 1 patient with unknown prior solid tumor, 1 patient with history of Hodgkin's lymphoma, and 1 additional patient with history of MPN who received chemotherapy and/or radiation therapy, with 3 patients having MGUS prior to development of MM. Notably, among this population, 70% were male. All patients developed MM following the treatment for a solid malignancy, lymphoma or MPN. The average amount of time from diagnosis of solid tumor, lymphoma or MPN to development of MM was 9.53 years (1-25 years). 50% of the individuals were White Americans, while the remaining 50% were African Americans. A significant majority (85%) of the population had a single primary solid tumor. Among the primary solid tumors, 45% were prostate cancer, 15% were breast cancer, and 10% were renal cell carcinoma (Figure 1). 5 patients received chemotherapy alone without documented prior radiation exposure while 13 had prior radiation exposure for prior solid malignancy. At time of diagnosis, 45% of patients had renal failure with 10% of patients having both renal failure and hypercalcemia. Within the t-MM subtypes, we discovered that 50% had IgA type, with 70% of these individuals exhibiting the IgA Kappa subtype. 45% of the individuals who developed t-MM had the IgG type, with 78% of them with IgG Kappa subtype. 15 (75%) of the patients received transplant and only 2 patients had confirmed relapse with 2 patients unknown and 2 additional patients who passed away prior to starting maintenance therapy following transplant. 4 patients are very early in their disease process and as of yet have not undergo transplant. 55% of our patients are presumed living. The OS for patients with t-MM was 1190 days compared to the average OS of 2326 days in our institutional MM database without t-MM. Conclusion Our analysis focused on patients diagnosed with MM who had a prior history of solid primary tumors, lymphoma or MPN treated with chemotherapy and/or radiotherapy in hopes of providing valuable insights into the characteristics and outcomes of individuals who developed MM following treatment for solid tumors, lymphoma or MPN. Our findings highlighted specific risk factors associated with t-MM in this population. Being male and having a history of prostate cancer was associated with higher incidence of development of t-MM. This is likely related to the radiation therapy associated with prostate cancer, as radiation exposure has been a known risk factor for the development of MM. We observed that the predominant subtype of MM among these individuals was IgA kappa, which diverges from the prevalent subtype, IgG kappa, observed in the general population. IgA is known to typically have more cytogenetic abnormalities and overall poorer prognosis, likely contributing to the difference in OS compared to patients with t-MM and MM not associated with prior malignancy. These insights shed light on the unique characteristics and potential influences of treatment-related MM in patients with prior malignancies or MPN.

Long Non-Coding RNA LINC00XPRR Is Upregulated in Newly Diagnosed Multiple Myeloma Promoting Poor Response to High-Dose Melphalan with Autologous Stem Cell Transplantation and Three-Drug Regimen

Abstract Multiple myeloma is an incurable malignant hematopoietic blood cancer. It develops from genetic disposition and abnormal plasma cells with over 30,000 new cases diagnosed in the United States every year. The 5-year relative survival rate for myeloma patients if diagnosed at early stage is 79.5%, however majority of patients are diagnosed once the cancer has already metastasized which decreases the 5-year relative survival rate to only 59%. The standard care for treating newly diagnosed multiple myeloma (NDMM) is three-drug induction therapy followed by high-dose melphalan (HDM) and autologous stem cell transplantation (ASCT). While there is no cure for multiple myeloma, diagnosis and standard therapy leads to prolonged remission and improved quality of life. Identifying genetic predictors for positive treatment responses and survival represents an unmet medical need. Long non-coding RNAs (lncRNAs) play important regulatory roles in promoting cancer growth and metastasis and due to their tissue-specificity they are becoming important players for development of personalized therapies. In addition, lncRNA-protein interactions have shown to be vital for biological processes associated with cancer, but currently there is a lack in our understanding of their role in initiation or progression of myeloma. Further, the roles of lncRNAs as biomarkers or targets for myeloma therapies have not yet been defined. In order to address these issues, we performed transcriptome sequencing from NDMM patients which identified 244 differentially expressed lncRNAs in poor responders (indicated as <24 months progression free survival (PFS)) to three-drug regimen, HDM and ASCT (n=38) compared to standard responders (indicated as >24 months PFS) to three-drug regimen, HDM and ASCT (n=78). Our analysis revealed LINC00XPRR (lncRNA00Xpoor responder-related) as one of the top-most upregulated novel lncRNA associated with poor responders to standard myeloma care. We next validated LINC00XPRR high expression in single cell RNA-sequencing data in NDMM patient samples. Multiplexed fluorescent RNA in situ hybridization (mFISH) analysisshowed that LINC00XPRR expression increased in melphalan treated myeloma cell lines and is highly expressed in bone marrow aspirates from patient samples. We further determined higher levels of LINC00XPRR in the cytoplasmic compartment of the cells compared to nuclear localization through QuPath analysis. LINC00XPRR mechanistically and epigenetically modulates cytotoxic and apoptotic phenotypes in myeloma cell lines when overexpressed in U266B1 and MM1.S cells or using RPMI8226 CRISPR/Cas9 knockdown system and in appropriate multiple myeloma-associated mouse models. Additionally, we used RNA immunoprecipitation (RIP) combined with qPCR to determined that LINC00XPRR directly interacts with CELF2, RNA-binding protein. Lastly, using target specific LINC00XPRR designed locked-nucleic acid antisense oligonucleotides (LNA ASOs) to knockdown LINC00XPRR, we saw an increase in apoptotic phenotypes indicating clinical potential of LNA ASOs-mediated LINC00XPRR knockdown. Overall, we are the first to identify lncRNAs associated with poor response in NDMM patients to standard three-drug induction chemotherapy followed by HDM and ASCT and show that LINC00XPRR enhanced expression may be both an important biomarker and target for future therapeutic interventions. Our current studies focus on the biology of lncRNAs in myeloma and the mechanism by which increased expression of LINC00XPRR is associated with poor outcomes in patients with NDMM receiving standard induction therapy, HDM and ASCT. Topics: multiple myeloma, long non-coding RNAs, LNA ASOs, RNA therapeutics, treatment response, RNA sequencing, apoptosis

Durable MRD and Imaging-Negative Remission ≥ 5 Years Post-Therapy Predicts Long-Term Progression-Free Survival in Multiple Myeloma Patients - a Single-Center Observational Study

Introduction With the advent of modern multiple myeloma (MM) therapies, a significant proportion of patients can achieve durable clinical remission. Minimal residual disease (MRD) reflects depth of response and is a powerful predictor of progression-free survival (PFS) (Munshi et al, JAMA Oncology 2017). Although sustained MRD negativity (defined by IMWG as MRD-negative on two occasions, minimum one-year apart) is associated with improved PFS, there is continued long-term relapse in these patients. A recent study reported a 10-year PFS of 88% in MM patients who are MRD-negative > 3 years after total therapy (Mohan et al, Blood Advances 2022). In the current era of regular MRD testing in MM care, we aimed to estimate the 10-year PFS of unselected MM patients who are MRD and imaging-negative ≥ 5 years after latest therapy, using our single-center data. Methods MRD testing was introduced for MM patients in remission at the Icahn School of Medicine at Mount Sinai (ISMMS) in 2017. Therefore, we systematically reviewed all patients with MRD results in the ISSMS MM registry between July 2017 and December 2022. Next-generation multi-parametric flow cytometry (NGF) was used to detect MRD from 2017 to 2021, with the addition of next-generation sequencing (NGS) from 2021 onwards. MRD-negativity was defined as <1 in 10 5 by NGF or <1 in 10 6 by NGS, and one representative MRD result per year was taken for analysis. Whole-body positron emission tomography (PETCT) and MRI were used to evaluate bone or extra-medullary (EM) disease. Detailed retrospective chart review of patients with sequential MRD-negative results was conducted to identify those with durable clinical, MRD, and imaging negative remission. PFS was evaluated using Kaplan-Meier curves and survival estimates. Results Between 2017 and 2022, 1703 MM patients underwent MRD testing at our center. Of these patients, we have so far identified 92 patients who were in CR, MRD and imaging-negative ≥ 5 years after the start of latest therapy. The estimated 10-year PFS in our cohort was 91.4% (95% CI [0.833 ,1]; Figure 1). With a median follow-up time of 5.9 years (4.6 to 12.6 years), the majority of our cohort (75%) were off-therapy at last follow-up with a median time-off-therapy of 2.5 years. In terms of baseline patient and disease characteristics (Table 1), 57.6% patients were white and 60.9% were male. Most patients were ISS stage I (51.1%) with standard-risk cytogenetics (78.2%) at diagnosis. A much smaller proportion of our cohort had any adverse cytogenetics, including gain1q (13%) and del17p or TP53 mutation (7.6%). The majority of patients were in their first remission after frontline therapy (52.2%), with 33.7% having received autologous stem cell transplant (ASCT) first-line. A smaller proportion of the cohort had received salvage treatment; 31.5% had received two lines, 16.3% had received ≥ 3 lines. Four patients relapsed in this cohort (4.3%) and one patient died of a non-MM related cause. Of the patients who progressed, 2 relapsed with isolated biochemical disease and 2 with predominantly EM disease. Both biochemical relapses were preceded by conversion to MRD positivity by NGF 6 months to 1 year prior, whereas those relapsing with EM disease remained MRD negative. Three patients had been off-treatment between 1 to 4 years prior to clinical relapse and the fourth patient progressed whilst on maintenance treatment. Conclusion We report single-institution MRD data on unselected MM patients who have durable MRD and imaging-negative remission for at least 5 years from their latest line of therapy. The estimated 10-year risk of disease-progression in our cohort is around 8.6%, with the majority of patients being off-therapy. The low rate of disease-progression at 10 years demonstrates that a proportion of MM patients do not suffer multiple relapses and may therefore be potential candidates for discontinuation of maintenance. From our data, these patients are more often ISS stage I, have standard risk cytogenetics and achieve deep responses to frontline therapy. A smaller proportion of patients are able to achieve this landmark even after salvage therapies. This may be increasingly possible in the era of monoclonal antibodies and T cell redirection therapies. Analysis of larger real-world MM cohorts is needed to elucidate whether sustained MRD-negative remission at 5 years can truly predict long-term disease-free survival and possible cure.

Racial Disparities in Autologous Stem Cell Transplant Utilization in Multiple Myeloma Persist Despite Referral to a Transplant Center

Introduction: Although the role of upfront autologous stem cell transplantation (ASCT) as an effective therapy for multiple myeloma (MM) is established in multiple clinical trials, it is well documented that non-Hispanic Black (NHB) and Hispanic individuals are less likely to receive ASCT for MM compared with age- and sex- matched non-Hispanic white (NHW) individuals. Methods: We conducted an institutional review of new patient consultations for MM at the Froedtert and Medical College of Wisconsin Cancer Center from 1/1/2012 to 12/31/2022, a tertiary medical center in the state of Wisconsin. We looked at the ASCT utilization rate by race and studied factors related to patients undergoing ASCT, including univariate and multivariate analyses. We utilized a logistic regression model to examine the likelihood of receiving ASCT. We considered the following covariates in the model building process: age, race, sex, cytogenetics, ISS stage, renal co-morbidity, year of consult, time from diagnosis to consult. A significance level of 0.1 was used for entry into the model. Race was self-reported by patients in the medical record. Results: We identified 1,221 MM consults from 2012 to 2022, of which 1,019 were NHW, 158 were NHB, and 44 were other minorities (“Other”) (Table 1). Median age at consult was 63, 66.1, and 61.8 years for NHBs, NHWs, and Other (p<0.1). All groups had similar cytogenetic risk, ISS staging, myeloma subtype, and time of diagnosis to consult. NHBs had longer median time from diagnosis to ASCT when compared to NHWs and Other, 6.4, 5.8, and 5.5 months, respectively (p=0.03). Median time from consult to ASCT was 4.3, 3.6, and 4.2 months for NHBs, NHWs, and Other (p=0.06). However, when comparing NHBs to NHWs, the difference between median time from consult to ASCT was significant (p=0.02). ASCT utilization rate was lowest in NHBs at 67.7% compared to Other at 72.7% and highest at 78.3% in NHWs (p=0.01) with trends over time shown in Figure 1. Multivariate analysis showed that NHWs were more likely to proceed to ASCT than NHBs (OR=2.698, 95% CI: 1.788-4.071, p=0.0021) while those with age ≥75 (OR=0.063, 95% CI: 0.044-0.09, p<.0001) and those with renal co-morbidity were less likely to proceed to ASCT (OR=0.523, 95% Cl: 0.36-0.761, p=0.0007). Conclusion: Consistent with prior studies, NHBs were younger at the time of their MM transplant consultation and had a longer median time from diagnosis and consultation to ASCT when compared to NHWs. Despite referral to a transplant center, NHBs still had lower ASCT utilization than NHW, indicating that simply access to a transplant center is not the only barrier driving ASCT utilization differences between NHBs and NHWs. Age ≥75 years and renal comorbidity were also associated with lower ASCT utilization per multivariate analysis. Future plans to further understand the role of social determinants of health and the underlying role of structural racism on access to ASCT in multiple myeloma will help identify targets for interventions to eliminate these barriers.

Prior Immune Effector Cell Therapy, Is Not Associated with an Increased Infectious Risk of Subsequent Bispecific Antibody Therapy for Multiple Myeloma

Introduction: Bispecific antibodies (BisAb) are effective therapies for relapsed myeloma yet associated with high risk of potentially severe infections. A prior study from our center did not associate any baseline characteristics with 90-day risk of infection. In clinical practice patients are already receiving sequential immune effector cell therapy (IET; BisAb or CAR T) it remains unclear whether this compounds the infectious risk. Herein, we examined whether a prior T-cell redirecting therapy predisposes to increased risk of infection during subsequent BisAb. Methods: This is a single-center, retrospective analysis of patients receiving BisAb therapy targeting B-cell maturation antigen (BCMA), G protein-coupled receptor family C group 5 member D (GPRC5D), or Fc receptor-homolog 5 (FCRH5) between January 2017 and February 2023. BisAb treatment was considered a treatment event (TE) such that if a patient received two different BisAbs, this would be considered two separate TEs. We obtained clinical data from before BisAb therapy start date: prior CAR T therapy, prior BisAb therapy, and infections 90 days before TE). Infection data during the full duration of BisAb therapy was also collected from the time of the first priming dose until 30 days after the last BisAb dose. For follow up for infections, we included any TE with at least one target dose of BisAb. We utilized two-sample proportion tests to analyze the differences in rates of infections between the BCMA and non-BCMA groups. Results: 91 patients and 123 TE episodes were identified. The TE episodes consisted of 40 with BCMA-directed therapy, 48 with GPRC5D-directed therapy, and 35 with FcRH5-directed therapy. Of these 123 treatment episodes, 26 TEs had 1 prior CAR T and 2 TEs had 2 prior CAR T and 28 TEs had received 1 prior BisAb and 6 TEs had received 2 prior BisAb. The most common infections were viral (46.1%) followed by bacterial (30.4%) and fungal (5.2%). We evaluated the relationship between prior IET (either CAR T or BisAb therapy) and incidence of infections during and up to 30 days BisAb (Table 1). In TEs with at least 1 prior CAR T the incidence of infections was: 62.5% (BCMA), 44% (GPRC5D) and 64% (FCRH5). For BCMA vs non-BCMA, the incidence of infections was 62.5% vs 55% (p=1.0). In TEs with at least 1 prior BisAb, the incidence of infections following subsequent BisAB was 50% (BCMA), 100% (GPRC5D) and 50% (FCRH5). For BCMA vs non-BCMA, the incidence of infections was 50% vs 65% (p=0.6). In TEs without prior IET, the incidence of infections was 50% (BCMA), 42.8% (GPRC5D), 75% (FCRH5). For BCMA vs non-BCMA, the incidence of infections was 50% VS 52.9% (p=1.0). On comparing these cohorts, we found no significant differences between the BCMA and non-BCMA BisAb therapy for following groups: prior BiSAb, prior CAR T, and the group without prior IET. We evaluated TEs with infections in the 90 days prior to BisAb and their relationship to infections during the first 90 days following BisAb therapy. The infection rate in the 90 days following BisAb therapy increased in TEs with earlier infection in the 90 days before BisAb priming dose from 30% to 43% for BCMA and 34.9% to 45.9% for non-BCMA BisAb. Conclusions: In this retrospective study we found similar rates of infection between groups with and without prior IET exposure. In patients who had infections during the 90 days before BisAb therapy, there was an increase in infection rate from baseline of 13% for BCMA BisAb and 11% from non-BCMA BisAb in the first 90 days after BisAb therapy. This is the largest cohort to report real-world data on infections following BisAb therapy after prior IET.

Resource Utilization Analysis of Two Algorithms for Hematopoietic Progenitor Cell Mobilization in Multiple Myeloma

Background: Autologous hematopoietic cell transplantation (HCT) is a standard of care after induction therapy for multiple myeloma (MM). A key step in this procedure is the mobilization and collection of hematopoietic progenitor cells (HPC). HPC mobilization typically uses G-CSF with or without the CXCR4 antagonist Plerixafor (P). Although P increases the effectiveness of mobilization and may reduce the number of apheresis sessions, it is associated with significantly increased cost per dose. There is significant heterogeneity to HPC collection algorithms across institutions and there is no clear standard. At the Cleveland Clinic, our HPC collection algorithm was updated in an effort to reduce the use and associated cost of P. The criteria for initiating P became more stringent and far fewer patients were initiated on P prior to their first session of HPC collection apheresis. Prior to October 2017, patients were initiated on P if any 1 of the following criteria were met: age>60 years, prior treatment with melphalan or other alkylating therapy, prior treatment with lenalidomide >2 cycles, extensive bone marrow disease, or platelet count <100K. Starting October 1, 2017, patients were preemptively initiated on P prior to first collection only if all 3 of the following criteria were met: age > 65 years, lenalidomide > 6 cycles, and platelet count < 100K. However, P was added after the first apheresis session if peripheral blood CD34+ cell count was <20/uL or HPC yield was unsatisfactory (<2 x 10^6 CD34+ cells). Furthermore, target CD34 yield was decreased from 5 to 4 x 10^6 CD34+ cells. Herein, we perform a comparative resource utilization analysis of HPC collection at our center. Methods: We retrospectively collected HPC mobilization data on patients undergoing HCT for MM from 1/12014 to 12/31/2020. We determined the number of P doses, number of apheresis sessions, and HPC collection totals per patient. Patient characteristics were summarized in median, interquartile range (IQR), or frequencies and percentages as appropriate. Fisher's exact test and Wilcoxon rank sum test were used to compare variables in patients undergoing mobilization prior to versus after 10/1/2017. All tests were two-sided and p-values of 0.05 or less were considered statistically significant. Results: 219 patients underwent HPC mobilization from 1/1/2014 to 9/30/2017 and 251 patients underwent mobilization after algorithm change from 10/1/2017 to 12/31/2020. As anticipated, the protocol change decreased the number of patients receiving P prior to their first apheresis session. 91% received P prior to the first apheresis session before the change while 14% received this initial dose of P after the change. Prior to the change 51% of patients completed collection in 1 day, 30% completed in 2 days, 12 % completed in 3 days, and 7% completed in 4 days or more. These figures changed to 20%, 41%, 23%, and 17% respectively following the change (p<0.0001) Median day 1 peripheral blood CD34 count decreased from 63 to 19 cells/uL while median day 1 CD34 collection yield fell from 4.8 to 1.46 x 10^6 after the change (p<0.0001) (Table 1). Total CD34 collection yield also decreased from a median of 6.64 to 4.6 x 10^6 CD34+ cells (p<0.0001). However, the target yield was decreased from 5 to 4 x 10^6 CD34+ cells as described above. The number of doses of P per patient decreased after the change (pre-change median dose / patient = 1, IQR = 1-2) (post-change median dose / patient = 1, IQR = 0-2) (p=0.004). The average number of apheresis sessions required per patient increased following the change (pre-change median apheresis sessions = 1, IQR = 1-2) (median post change apheresis sessions = 2, IQR = 2-3) (p<0.0001) (Figure 1). Conclusion: Changes to our institutional HPC mobilization algorithm led to a substantial decrease in the use of preemptive P prior to day 1 of collection by design. The resulting decrease in total doses of P per patient was smaller than expected, although still statistically significant. However, this was offset by a concomitant significant increase in the number of apheresis sessions required per patient despite a lower collection target. Based on these results, a cost analysis is ongoing in order to re-evaluate our institutional algorithm.

Hdp-101, an Anti-BCMA Antibody-Drug Conjugate with a Novel Payload Amanitin in Patients with Relapsed Multiple Myeloma, Initial Findings of the First in Human Study

Introduction Several antibody-drug conjugates (ADCs) are currently being evaluated in clinical trials in a variety of malignancies. The vast majority of these are based on a few toxic compounds, largely limited to microtubule- or DNA-targeting toxins which impact proliferating cells and have limited efficacy in diseases with a low proliferative fraction such as multiple myeloma. Thus, new compounds with alternative modes of action and the ability to actively induce cell death in non-proliferating tumor cells could enhance the therapeutic potential of ADCs. We are currently developing amanitin based ADCs. Amanitin specifically inhibits RNA polymerase II thereby inhibiting the cellular transcription process at very low concentrations irrespective of the proliferation status of the target cell. Subsequently tumor cells enter apoptosis and are eliminated. HDP-101 an Amanitin based antibody drug conjugate HDP-101 is a new ADC targeting BCMA carrying a synthetic version of amanitin as a payload. In vitro cytotoxic potency of HDP-101 was demonstrated on BCMA-positive myeloma cell lines, as well as on non-proliferating primary CD138+ cells isolated from patients with refractory myeloma The cytotoxic effects of HDP-101 were seen even in non-proliferating myeloma cells with low BCMA density. Toxicity was observed neither in non-BCMA expressing control cells nor in myeloma cells exposed to an amanitin-loaded non-target control antibody. In murine xenograft models of human myeloma, HDP-101 caused dose-dependent tumor regression including complete remissions after a single dose in subcutaneous and as well as in disseminated models. Safety profiling in Cynomolgus monkeys revealed a good therapeutic index after repeated dosing. Our non-clinical studies concluded that this amanitin-based ADC is a novel promising approach in the therapy of multiple myeloma to overcome drug resistance and improve patient outcome. HDP-101-01 Clinical study HDP-101-01 is a first-in-human, open label, non-randomized, multicenter, phase 1/2a trial with HDP-101 in patients with myeloma whose disease has progressed. The aim of the Phase 1 dose escalation part is to determine the Maximum Tolerated Dose and/or establish the Recommended Phase 2 Dose. The primary objective of the phase 2 dose expansion phase is to assess the preliminary anti-tumor activity of HDP-101. An adaptive Bayesian logistic regression model with overdose control principle is used to guide the dose escalation steps. An Interim Analysis is planned after each cohort is completed. The design of the study ensures a safe and adaptive dose escalation to reach a potential clinical benefit in a patient who have limited or no therapeutic options. HDP-101-01 Study progress The study started enrollment in February, 2022. As of 10th of July, 2023, eight (2 female and 6 male) patients were dosed in 3 consecutive dose cohorts. The median age of the patients was 70 years, ranging between 50 and 80. All 8 patients were heavily pre-treated and multidrug-resistant. The median previous lines of treatment were 7 (5 to 15). Table 1 HDP-101-01 Results Seven of 8 patients were evaluable for dose limiting toxicities (DLTs) in the first 3 treatment cohorts. The initial 3 cohorts were well tolerated, without any DLTs, there were no signs of liver and kidney toxicity, no infusion reaction was detected. No reports of keratopathy or visual acuity loss were observed. Free payload was not detected in any of the available pharmacokinetic samples. Based on the limited data, the PK of HDP-101 was in line with our expectation based on the preclinical observations, exposure to HDP-101 is dose proportional. Anti-drug antibody (ADA) was not detected and there's no sign of immunogenicity. Objective responses were not reported in these initial cohorts however in Cohort #3 (60μg/Kg) there was one patient ongoing after 8 cycles of treatment with SD. This patient had a slow decrease in M-protein levels. Image 1 The initial dose cohorts showed good tolerability in late stage relapsed and/or refractory multiple myeloma patients. The study continues to enroll patients to higher dose cohorts at selected sites in the US, Germany, Poland and Hungary. Currently the study is enrolling patients in Cohort #4 at the dose of 80μg/Kg. Additional updates will be provided at the 2023 ASH Annual Meeting.

Optimal, Off-the-Shelf, CAR-iNKT Cell Platform-Based Immunotherapy for Multiple Myeloma

Autologous CAR-T cell immunotherapy targeting BCMA confers significantly improved progression free survival as compared to standard-of-care in relapsed/refractory multiple myeloma. However, despite >50% complete remission rates, patients continue to relapse. Of note, in current licenced BCMA CAR-T products, 4-1BB comprises the co-stimulatory domain of a 2 nd generation CAR. Expression of CD1d by myeloma plasma cells (PC) lends itself to immunotherapy with the CD1d-restricted, glycolipid-reactive iNKT cells which can be deployed ‘off-the-shelf’ without causing aGVHD. We hypothesised that ‘off-the-shelf’ anti-BCMA CAR-iNKT would be an alternative platform for the treatment of MM. We sought to identify the CAR design that would function optimally in conjunction with iNKT cells. To this end, we designed several different second (28z, 4-1BBz, OX40z) and third (28z-4-1BBz, 28z-OX40z) generation BCMA CARs and tested their anti-myeloma activity. Lentiviral transduction efficiency of iNKT cells was comparable for all five CARs and all five CAR-iNKT were able to kill two BCMA-expressing but not BCMA knock out myeloma cell lines in short- and long-term cytotoxicity assays. The level of cytotoxicity varied with donor, timing of the assay and cell lines, with the only consistent pattern seen with the 3 rdgeneration CD28-4-1BB CAR. This showed the least reactivity at low E:T ratios in both 4 and 16hrs assays for both donors tested. Testing cytotoxicity against primary myeloma PC (n=3) showed that the 3 rd gen CARs were less active than the three 2 nd gen counterparts. Short term proliferation assays using Incucyte Zoom demonstrated a higher proliferative potential of CD28z CAR-iNKT cells with CD28z-4-1BBz CAR showing the least proliferation. In a longer term 35-day assay, following stimulation with the iNKT cell ligand aGalCer, we found that CD28z CAR-iNKT cells showed the highest expansion. To further probe functional differences between the five CARs, we tested the corresponding CAR-iNKT cells in acoustic force avidity assays. These highlighted CD28z CAR-iNKT cells as having the highest avidity amongst CAR-iNKT cells with untransduced iNKT cells the lowest (Fig. 1A ). Using confocal microscopy, we explored how these differences in avidity impacted the ability of CAR-iNKT cells to shape the immune synapse (IS) and engage the cytolytic apparatus. We found that while conjugates of untransduced iNKT cells with myeloma cells all fell in the distal centrosome group, of the five CARs, CD28z and 4-1BBz CAR-iNKT conjugates recorded the highest and the lowest respectively proximal and docked conjugates, i.e., those signifying imminent or active discharge of the cytolytic granules. To delve further into the mechanism(s) of higher avidity and ability to established IS by CD28z CAR-iNKT, we compared the transcriptomes of CD28z vs 4-1BBz CAR-iNKT from three donors. We found 64 genes differentially expressed (14 up- and 50 down-regulated in CD28z CAR-iNKT, padj<0.05). Amongst the upregulated genes we identified, currently investigating genes that encode proteins that were previously implicated in the strength and duration of IS. To investigate the in vivo correlates of these functional assays, we deployed a xenograft myeloma model using MM1.S luciferase-expressing cells (Fig. 1B ). All five CAR iNKT cells were i.v transferred to myeloma-bearing mice at the limiting dose of 10 6 CAR+ cells/mouse and disease burden was monitored by bioluminescence. In line with the functional assays, we found that mice receiving CD28z CAR-iNKT cells demonstrated the lowest disease burden and survived the longest (p<0.01). While disease burden and survival of animals receiving CD28z-4-1BBz CAR-iNKT was the same as controls, 4-1BBz CAR-iNKT-treated mice showed significantly higher disease burden and shortened survival as compared to CD28z CAR-iNKT. Further disease burden assessment by flow-cytometry at sacrifice showed that CD28z CAR-iNKT-treated animals had the lowest (p<0.01) frequency of myeloma cells in their peripheral blood, bone marrow and spleen. Finally, in ongoing xenograft assays, we find that compared to 10 6 28z BCMA CAR-iNKT and 5x10 6 untransduced iNKT, 5x10 6 28z BCMA CAR-iNKT cells maintain better disease control (p=0.008, day7). We conclude that CD28z by enhancing effector-target avidity and strength of IS constitutes the optimal CAR design for clinical development of CAR-iNKT therapy of multiple myeloma.

Artificial Intelligence of the 2-D and 3-D Bone Marrow Microenvironment to Identify Cytogenetic Subtypes of Multiple Myeloma

Multiple myeloma is the second most common hematologic malignancy with an annual incidence of 35,000 new cases every year in the U.S. Due to improved access to effective therapies, overall survival has improved from 1-3 years to over 10-20 years. However, there is not yet any available curative therapy for multiple myeloma and most patients will suffer from recurrent relapses, with an unmet clinical need for novel therapies. The interplay between the bone marrow microenvironment and malignant plasma cells is critically important to better understand pathogenesis of the disease, to predict response to therapy (in particular, immunotherapy), and to identify novel drug targets. Leveraging the power of computer vision, we are developing novel machine learning algorithms to label and characterize individual cells from digitalized H&E and CD138 stained plasma cells in bone marrow core biopsies. A total of 188 patients were included in this analysis: 157 with multiple myeloma, 6 with monoclonal gammopathy of undetermined significance, 17 with smoldering multiple myeloma, and 6 with primary amyloidosis. The deep learning framework to build our artificial intelligence (AI) models is implemented by Pytorch. Using the surface creation tool from the Imaris 10.0.1 software, we are able to measure pixel intensity, area, sphericity, and other cellular features that are then correlated with myeloma clinical outcomes as well as defined cytogenetic subtypes of multiple myeloma in 2-D. As a preliminary step in this project we analyzed the IMARIS software's ability to accurately determine the number of plasma cells compared with clinical standards through a linear regression plot with strong correlation (R 2=0.9). In addition, we rendered the 3-D microenvironment by combining more than 60 sequential formalin-fixed paraffin embedded (FFPE) tissue block bone marrow biopsy samples sliced at a thickness of 4 microns. Through utilizing the surface creation tool on the 2-D images within the 3-D model, we can show distance measurements between plasma cells and other features of the microenvironment such as adipocytes. This insight allows us to better understand and control the immune microenvironment through observing the various 2-D tissue slices within the 3-D model. Furthermore, we have assessed whether multiple myeloma digital images are able to detect translocation [t(11;14)] multiple myeloma subtype, which is present in 15% to 24% of patients, shown to predict treatment response to Bcl-2 inhibitor therapy (such as venetoclax). A small cohort (n=25) of patients with t(11;14) have been compared to non-t(11;14) cases using the machine learning model and preliminary data indicate that there is a correlation between digital images and multiple myeloma subtype. In the future, we anticipate our AI model will improve the accuracy of diagnostic methods while reducing costs and offering deeper translational insights that will predict drug response in vivo.

Identification of LPAR1/LPAR5 As Novel GPCR Partners of GPRC5D for the Efficient CAR-T Therapy of Multiple Myeloma

Introduction: The emergence of chimeric antigen receptor T-cell (CAR T) therapy has brought hope to the treatment of refractory or relapsed multiple myeloma (RRMM). GPRC5D is an orphan G protein-coupled receptor (GPCR), which has a seven-transmembrane structure, and really high expression in multiple myeloma cells. So that CAR-T cells that target GPRC5D and this has been validated in a phase I/II clinical trials with encouraging clinical efficacy and manageable safety profiles of anti-GPRC5D CAR T-cell therapy for RRMM. It is of great clinical significance to find ways to further improve the efficacy of anti-GPRC5D CAR T-cell therapy. Methods: Our previous study based on a single-cell transcriptome analysis revealed a strong co-expression correlation of GPRC5D and lysophosphatidic acid receptor 1/5 (LPAR1/5). To further confirm the interaction with GPRC5D, LPAR1 and LPAR5 were tested by co-immunoprecipitation (co-IP) screening, bimolecular fluorescence complementation (BiFC), multiple pharmacological ELISA assays, and flow cytometry (FACS) experiments. The MM cell lines (RPMI 8226, L363 and KMS11) were treated with activator or antagonists of LPAR1 or LPAR5, and then co-cultured with anti-GPRC5D CAR T-cells. The cytotoxicity and cytokine analysis were determined by luciferase-base killing assay and FACS. Results: The results showed the obvious interactions between LPAR1/LPAR5 and GPRC5D from co-IP and BiFC assays, strongly supported the prediction from the bioinformatic analysis. The heterodimers of GPRC5D and LPAR1/LPAR5 influenced membrane expression of GPRC5D to different degrees based on ELISA assay. The HEK293T surface expression of GPRC5D was reduced in the presence of LPA receptors activator (1-Oleoyl lysophosphatidic acid, 1-Oleoyl-LPA), which indicated that both LPAR1 and LPAR5 had trafficking effect on GPRC5D. To further confirm these effects, the MM cell lines, RPMI 8226, L363 and KMS11, were cultured with 1-Oleoyl-LPA or combining antagonists of LPAR1 (AM966) or LPAR5 (TC LPA5 4). The cell-surface change of GPRC5D expression had no statistical difference when MM cells were only treated with 1-Oleoyl-LPA. However, the surface expression of GPRC5D of MM cell lines was increased with the combination of LPAR1/LPAR5 activator and antagonists by FACS detection To further investigate the above effects could promote the efficacy of anti-GPRC5D CAR T-cell therapy, MM cell lines and anti-GPRC5D CAR T-cells were co-cultured, with or without the treatment of activator and antagonists. The results showed that the CAR T-cell killing capacity to MM cell lines was reduced after the treatment of 1-Oleoyl-LPA, while its killing capacity was significantly recovered after performing a combination of 1-Oleoyl-LPA and AM966/TC LPA5 4. Notably, the levels of IL-2 and TNF-α in 1-Oleoyl-LPA groups were lower than the mock groups, and increased after the combination of AM966/TC LPA5 4. But, IFN-γ did not display significant difference. These data indicated that the regulation of LPAR1 and LPAR5 function might have enhance the efficacity in anti-GPRC5D CAR T-cell therapy. Conclusions: Our study provides hint evidence that LPAR1 and LPAR5 increase the MM cell-surface expression of GPRC5D and improve the efficacy of anti-GPRC5D CAR T-cell therapy, which might be as novel GPCR partners of GPRC5D for the efficient CAR-T therapy of multiple myeloma.