Chesi, Marta

ABSTRACT Chimeric antigen receptor T-cell (CART) and T-cell engager (TCE) therapies targeting B-cell maturation antigen (BCMA) are transforming the treatment landscape for relapsed multiple myeloma (MM). However, despite impressive initial response rates, most patients eventually relapse. To investigate this unmet medical need, we applied whole-genome sequencing (WGS) to MM cells from cohorts of 102 relapsed patients treated with anti-BCMA CART and TCE therapies. Several genomic alterations were associated with clinical outcomes, particularly primary refractoriness, including high genomic complexity and mutations in genes regulating plasma cell identity, which predicted resistance to therapy. Single-cell RNA sequencing further revealed that MM cells from refractory patients exhibited high proliferation signatures and reduced expression of TNFRSF17 (encoding BCMA), while were less enriched for plasma cell–associated transcriptional programs, a phenomenon we term “plasma cell identity escape.” This profile was strongly associated with immune dysregulation of CD8 T cells including increased activation and exhaustion. This evolution of MM toward a more proliferative and lineage-divergent state, refractory to the anti-BCMA T-cell redirecting therapies, was functionally validated in preclinical MM mouse models. Collectively, our results comprehensively define the cellular and molecular mechanisms underlying primary resistance to anti-BCMA therapies.

Abstract Introduction High disease burden has been associated with reduced efficacy and resistance to BCMA CAR-T therapy in relapsed/refractory multiple myeloma (van de Donk, Blood Cancer Discov, 2021; Martin, J Clin Oncol, 2023). While CAR-T cells can still induce remissions in these patients, outcomes are often worse compared to those with a lower tumor load. In addition to tumor debulking, novel strategies are needed to improve responses and durability of CAR-T cells in these patients. We previously demonstrated that immunomodulatory drugs (IMiDs) improve in vivo responses to T cell engagers (TCE) in high tumor burden setting by boosting T cell activation (Meermeier, Blood Cancer Discov, 2021; Meermeier, Blood, in press). IMiDs have also been shown to promote CAR-T cell antitumor activity in moderate to low tumor burden conditions (E:T ratios ≥1:1) (Yan, J Transl Med, 2024; Wang, Clin Cancer Res, 2018). We hypothesized that combining CAR-T with an IMiD may improve CAR-T function in high tumor burden setting. Methods We generated murine anti-BCMA.CD28 CAR-T cells by transducing Con-A stimulated T lymphocytes extracted from hCRBN+ mice, expanded with IL-7 and IL-15 to express a memory phenotype. We then tested CAR-T, or untransduced T cells, ability to kill in vitro IMiD resistant, hCRBN-, Vk*MYC myeloma cells in low (E:T 1:1) and high (E:T 1:≥5) tumor burden, with and without pomalidomide (POM) and IL-2. Tumor killing and T cell phenotype were assessed by flow cytometry at 24-72 hours. This controlled system allowed us to focus on putative CAR-T cell intrinsic impact of IMiDs. Results In low tumor burden setting, CAR-T effectively killed tumor at all timepoints, with or without POM; addition of POM increased T cell number, confirming that IMiDs support CAR-T expansion. Unexpectedly, in high tumor burden (E:T 1:≥5), adding POM diminished CAR-T mediated killing over time and hindered CAR-T expansion. Moreover, combination with POM decreased granzyme B, while increased expression of exhaustion marker TOX, ultimately leading to CAR-T apoptosis. This phenotype was further intensified by increasing concentrations of IL-2 in the co-culture, resulting in increased expression of CAR-T expressing LAG-3, TIM-3, and TIGIT, suggesting that CAR-T cells may be overactivated by POM and IL-2. Our manufacturing protocol preferentially expands memory CAR-T cells, favored for their durability, proliferative capacity, and considered to rapidly expand upon encounter with antigen. We reasoned that CAR-T are already sufficiently activated by CAR/CD3z stimulation, especially with a CD28 co-stimulatory domain (versus 4-1BB), known promote rapid, effector memory expansion upon activation (Kawalekar, Immunity, 2016). Activation is intensified by high tumor burden where antigen is highly abundant. We hypothesize under intense, sustained antigen exposure in high tumor burden, the addition of POM paradoxically triggers activation induced cell death (AICD). Conclusion Although it is well accepted that POM can boost T cell function and proliferation, we demonstrate that in high tumor burden, POM drives memory CAR-T to increase exhaustion markers and apoptotic cell death by overactivation, further exacerbated by exogenous IL-2. Interestingly, we have shown opposite effects of preclinically combining POM with TCE therapy in high tumor burden, based on dosing schedule. Concomitant administration transiently boosted T cell activation improving response rates but rapidly led to T cell exhaustion (Meermeier, Blood Cancer Discov, 2021). In contrast, IMiD pretreatment in the context of step-up dosing TCE administration improved T cell fitness and longevity (Meermeier, Blood, in press).The effect of IMiDs on T cells transcriptional program is likely to differ depending on differentiation stage and mode of T cell activation. Memory cells have more sensitive TCR signaling (Kumar, Immunity, 2011) and can be hyperactivated, leading to AICD. Thus, timing of IMiD administration, when tumor burden is low, may be a critical consideration as different T cell subsets require different levels of activation. Conversely, TCE provide only initial activation via TCR signaling, therefore POM may help in providing necessary co-stimulation in this setting. Future studies will evaluate different co-stimulatory domains and test in vivo effects of POM on CAR-T expansion and persistence, particularly as CAR-T cells diminish over time as tumor burden increases.

Abstract Overview: The emergence of effective immune therapies for multiple myeloma has highlighted the importance of understanding the roles of less abundant components within the tumor microenvironment. Single-cell analyses provide snapshots of the functional relationships of individual cells at precise moments in time, as well as insight into the overall cellular composition. By integrating single-cell datasets from multiple studies, we improve our ability to identify trends within rare cell populations, such as macrophages, and explore their clinical implications. We investigate human data here because our prior research of murine models indicated that macrophages expressing C1q, particularly those co-expressing Fcgr4, tend to correlate positively with increased tumor burden. Methods: We combined 56 human bone marrow samples – primarily collected at baseline, post-induction, or post-ASCT – with three publicly available data sets: GSE210079 (post CAR-T bone marrow microenvironment); GSE223060 (single-cell analysis of the MMRF cohort); and GSE271107 (disease progression from healthy donors and MGUS to SMM and NDMM). These data sets were integrated using Seurat to generate robust cellular clusters from over 300,000 cells across 117 samples. We validated our single-cell findings with bulk Affymetrix, whole bone marrow gene expression data (collected at baseline and post-baseline) from the Total Therapy clinical trials (GSE136324). Results: A distinct cluster of C1QA/B/C-expressing macrophages was consistently identified in all single-cell cohorts. These cells co-express FCER1G, CSF1R, and CD68 (markers also expressed in murine C1Q macrophages) and typically cluster adjacent to FCGR3A-expressing monocytes. Within the disease progression cohort, C1Q macrophages were found in all phases of progression: healthy donors, MGUS, SMM, and NDMM. However, C1Q macrophages in progressive disease (SMM and NDMM) have distinct expression features including lower levels of MRC1 and higher levels of APOBEC3A and ISG15 – genes linked to inflammatory responses. Among 11 post-induction samples submitted to lenalidomide maintenance, five with shorter responses displayed elevated expression of APOBEC3A and ISG15 in C1Q-expressing macrophages compared to six samples with longer responses. In a cohort of 22 samples 100 days post-ASCT, seven poor responders showed both an increased frequency of C1Q macrophages and elevated APOBEC3A expression compared to good responders. The MMRF cohort indicated that higher baseline frequencies of C1Q macrophages often preceded rapid disease progression (e.g. MMRF_1720, MMRF_1641). Anecdotal evidence from the CAR-T therapy cohort suggests that expansion of C1Q macrophages could serve as an early warning for tumor recurrence; sample 16 showed retention and expansion of C1Q macrophages 28 days post-CAR-T, that was subsequently followed by increased tumor cell counts at 3 months post-therapy. Analysis of bulk, whole bone marrow gene expression revealed that elevated levels of C1Q genes correlated with poorer outcomes, particularly in post-baseline samples. Multivariate Cox survival analysis revealed that high C1Q expression independently predicted inferior overall survival in post-baseline samples after adjusting for age, ISS stage, high levels of tumor-associated markers (SDC1, JCHAIN, DERL3), and high-risk UAMS7 subtypes (MF, MS, PR). Moreover, post-baseline GEP70 risk scores (from matched bone marrow aspirates) positively correlated with elevated C1Q expression in whole bone marrow samples. Conclusion: Both single-cell and bulk analyses indicate that the persistence of immunosuppressive C1Q macrophages following therapy is associated with inferior clinical outcomes. Although the precise mechanisms are yet to be fully understood, the close proximity of similar macrophages to tumor cells–as observed in other cancers–likely also occurs in myeloma and reflects the crucial relationship between tumor cells and macrophages within the bone marrow. While C1Q macrophages are typically rare in individual samples, analysis of combined cohorts is building evidence of their crucial role in immune cross-talk and therapeutic resistance mechanisms.

Abstract VSV-IFNβ-NIS (VV1) is a Vesicular Stomatitis Virus (VSV) that encodes the interferon beta (IFNβ) and the iodide symporter (NIS) gene. The IFNβ gene confers tumor selectivity and enhances host antitumor immunity while the NIS gene acts as an imaging biomarker, allowing noninvasive imaging of viral infection sites using PET/SPECT. Multiple myeloma (MM) is a cancer of plasma cells and poses a challenge for treatment due to its biological heterogeneity. As MM usually relapses and is refractory, current therapies face the issue of drug resistance and patients may have to turn to novel therapies. Therefore, we set out to understand the activity of VSV which could be a new approach for patients with relapsed refractory MM (RRMM). Our study utilized two unique bortezomib resistant Vk*MYC transplantable models, Vk12598 and Vk12653, with distinct genomic features. Upon re-transplantation of splenocytes from these mice, flow cytometry revealed that Vk12598 and Vk12653 mice had high MM populations in the spleen and bone marrow. To assess VV1 efficacy in tumor bearing mice, we performed a VV1 dose escalation study, showing efficacy in delaying MM development and prolonging survival in both mice models, with Vk12598 being the more responsive model. Later studies also showed that VV1 induces tumor remission in the Vk12598 model when treatment was initiated at low tumor burden (gamma/albumin ratio ∼0.2). IFNβ levels increased significantly post 24-72h of VV1 treatment, indicating targeted infection and replication of the virus in MM cells. However, high doses of VV1 led to toxicity, as observed from early mortality and weight loss. To determine the sites of MM metastasis and VV1 infection, timepoint harvests were carried out. Hematoxylin and eosin and immunostainings showed the onset of MM of Vk*MYC in the bone marrow and spleen first, then later in the liver. Immunofluorescent staining confirmed that the higher the tumor burden observed, the more VV1 was present in the tissues. To enhance VV1 efficacy, we performed in vivo combination therapies of VV1 with anti-PD-1, an inhibitor of PD-1 which is overexpressed on MM patient T cells; and bortezomib, which inhibits NF-κB which in turn prevents IFN pathway protein transcription, potentially enhancing the replication of VV1 virus. In Vk12598 mice, VV1 combined with both drugs induced tumor regression and doubled the median survival time compared to treatment with VV1 only or VV1 coupled with either of the drugs individually. Addition of VV1 to either anti-PD-1 or bortezomib alone also increased survival time significantly. Studies are still ongoing to understand the mechanisms of VSV and its combination treatments in Vk*MYC models. This translational study will allow us to gain the knowledge needed to refine VSV treatments and provide novel therapy combinations to improve MM patient survival. Citation Format: Yi Ying Ma, Lianwen Zhang, Rebecca A. Nace, Marta Chesi, Stephen J. Russell, Kah Whye Peng. Harnessing oncolytic virus VSV and combination therapy to advance treatments for relapsed refractory multiple myeloma using Vk*MYC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 940.

Overview: Despite advances in therapies targeting the tumor-intrinsic aspects of the disease, multiple myeloma (MM) remains functionally incurable. Given that new alternatives such as T cell redirected therapy and monoclonal antibodies engage ADCC and CDC by leveraging other elements of the immune system, we believe that deeper studies of the tumor microenvironment are warranted. In particular, we believe that certain macrophages are associated with tumor progression, serve as markers of future relapses, are immunosuppressive, and reduce the effectiveness of otherwise successful therapies. Methods: We performed 5' scRNAseq analysis of 189,025 cells collected from spleen or bone marrow of 4 wild type, 14 de novo, and 40 tumor bearing Vk*MYC mice treated with vehicle or effective anti-MM therapies. Discoveries were cross-referenced against a set of 45,626 human bone marrow cells collected from 35 samples at different stages of disease and treatment. We experimentally recreated in vitro the cross-talk between MM cells and macrophages by co-culturing bone marrow derived macrophages (BMDM) with MM cells or their conditioned medium, and correlated changes in gene expression as well as the ability of polarized BMDM to inhibit CAR T mediated killing of MM cells in vitro. Results: Gene expression analysis of murine in vivo C1q macrophages and in vitro polarized BMDM revealed a common signature identified in lipid-associated macrophages and Kupffer cell-like macrophages1 (high expression of Fabp5, ApoE, Cd63, C1qa/b/c, Cd163, Cd5l, Vcam1, Fcgr4, Folr2, Maf) which is associated with immunosuppression and disease progression. Ligand-receptor analysis via LIANA identified C1qb ligand in macrophages with C1qbp in tumor among the top interactions in both human and murine single cell analyses. C1qbp is a crucial element of cell adhesion and metastasis,2 and this link between tumor and macrophage can severely limit effective therapy: BMDM following stimulation/polarization with MM lines, but not naïve/unstimulated BMDM, strongly inhibit anti-BCMA CAR T in vitro cytotoxic activity. In total, a complement-related connection between tumor and macrophage is capable of directing an immunosuppressive, chemokine response that imparts resistance to therapy. While effective therapies reduce tumor and C1q expressing macrophage populations,3 retention of C1q expressing cells may be associated with future relapses. Presence of a specific subset of C1Q expressing cells, even in very low numbers, in human samples following ASCT was associated with early relapse (6 of 8 good responding samples had zero C1Q+ cells while 12 of 16 poor responding samples had 1 or more C1Q+ cells-Fisher test p-value < 0.05). In addition to high expression of C1QA/B/C, these cells are also highly upregulated in TYROBP (known to relate to macrophage polarization4 and found in a spatial transcriptomic analysis of cells at the periphery of MM and its environment5) as well as FCER1G (known to relate to macrophage infiltration6). Conclusion: As advanced therapies manipulate elements of the immune microenvironment to impart effective treatment, the connection between tumor and macrophage should not be overlooked since macrophages likely help create an immunosuppressive niche that assists in disease growth and eventual revival. 1. Ma, R.-Y., Black, A. & Qian, B.-Z. Macrophage diversity in cancer revisited in the era of single-cell omics. Trends Immunol. 43, 546-563 (2022). 2. Wang, Y. et al. C1QBP suppresses cell adhesion and metastasis of renal carcinoma cells. Sci. Rep. 7, 999 (2017). 3. Stein, C. K., Meermeier, E., Welsh, S. J., Chesi, M. & Bergsagel, P. L. Key Subsets of Immune Cells in Bone Marrow Myeloid Compartment Indicate Degree of Tumor Infiltration and Avenues for Successful Immunotherapy in Multiple Myeloma. Blood 140, 7082-7083 (2022). 4. Liang, T. et al. TYROBP, TLR4 and ITGAM regulated macrophages polarization and immune checkpoints expression in osteosarcoma. Sci. Rep. 11, 19315 (2021). 5. Sudupe, L. et al. Spatial Transcriptomics Unveils Novel Potential Mechanisms of Disease in a MI cγ1 Multiple Myeloma in vivo Model. Blood 142, 4076 (2023). 6. Dong, K. et al. FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity. BMC Cancer 22, 140 (2022).

Introduction/Background: Bispecific T cell engagers (TCE) targeting BCMA and CD3 induce deep hematologic responses in approximately 60% of heavily pre-treated multiple myeloma (MM) patients. There is an unmet need to overcome primary resistance, as this off-the-shelf therapy is readily available to patients with aggressive disease relapse and has the potential to be delivered in the community. We and others found that high tumor burden leads to primary resistance to TCE 1,2,3,4 and that combination with immunomodulatory drugs (IMiDs) transiently improves responses yet exacerbates T cell exhaustion2. Here, we present preclinical strategies to overcome primary resistance which are tolerable and provide more durable responses specifically for the high tumor burden setting. Methods: We evaluated response and survival to a murine surrogate BCMA/CD3 TCE in the Vk*MYC immunocompetent mouse model of MM expressing human CRBN to investigate tumor burden as a mechanism underpinning primary resistance. We evaluated dosing methods and combination strategies with IMiDs or novel CELMoDs, checkpoint inhibitors, and dexamethasone to improve response rates while maintaining a manageable safety profile. Results: We investigated primary resistance in an expanded panel of Vk*MYC models reflecting differing genomic heterogeneity and confirmed that a threshold of tumor burden limits T cell activation and correlates with resistance to TCE. BCMA is cleaved from the tumor cell surface by gamma-secretase and thus, high density of shed BCMA may act as a sponge to BCMA TCE. However, we found that administration of gamma-secretase inhibitors, while stabilizing BCMA expression on MM cells, is not sufficient to overcome high tumor burden-driven resistance to TCE. We previously reported the combinatorial activity of BCMA TCE with IMiDs and revealed a role for tumor-extrinsic effects of IMiDs in allowing mice with a high tumor burden to transiently respond to BCMA-TCE before T cell exhaustion prevailed2. To build on that combination strategy, we first hypothesized that the addition of a PD1 checkpoint inhibitor could reduce T cell exhaustion that occurs in response the BCMA-TCE in the resistant setting. We found that this triple combination provides more persistent activation and expansion of cytolytic T cells and sustains remission but leads to toxic cytokine release syndrome and early mortality in half of the cohort. Next, to address the acute toxicity from the hyperactivated T cell response and reduce tumor burden, we combined the triple combination with dexamethasone (Dex). To our surprise, concurrent Dex, even in the absence of IMiDs/CELMoDs, boosts responses to TCE. We observe that Dex moderates T cell proliferation in response to the TCE and leads to a moderate sustained cytokine production (IFN-γ, TNF, and IL-2R) systemically and in the bone marrow compared with a high transient burst seen with TCE alone or the concurrent treatment with IMiD + PD1 blockade. We determined that incremental step-up TCE dosing allows the combination of IMiDs or CELMoDs (iberdomide or mezigdomide) with Dex to be completely tolerated. To investigate whether delivering IMiD or CELMoDs before or during TCE administration altered efficacy, we tested pre-treatment with IMiDs/CELMoDs + Dex compared with concurrent dosing and found pre-treatment led to superior durable responses. Pre-treatment with iberdomide + Dex followed by step-up dosed TCE generated 100% response rate and the highest overall survival in subjects bearing a high tumor burden. This was accompanied by a more favorable T cell profiling over time, with limited expansion of regulatory T cells and reduced accumulation of exhausted T cells. Conclusion: Modelling the dosing and combination strategies of a BCMA-TCE in our immunocompetent system has allowed us to understand the dynamic changes in T cell fitness that are required for effective, tolerable, and durable TCE therapy for MM. We illustrate how improvements to TCE therapy mitigate T cell exhaustion and overcome primary resistance, and importantly, suggest a strong immunotherapeutic strategy for MM patients with challenging-to-treat actively progressing disease. 1. Lee et al., Nat Med 29, 2295-2306 (2023) 2. Meermeier et al. Blood Cancer Discov. 2021 Jul;2(4):354-369. 3. Rees et al. Blood Cancer J. 2024 Jul 23;14(1):122. 4. Girgis et al. Blood Adv. 2023 Feb 28;7(4):644-648

Primary resistance to BCMA-directed bispecific antibodies (BCMA-BsAb) is a major clinical hurdle despite their outstanding efficacy for relapsed and refractory multiple myeloma (MM). Primary resistance has been linked clinically to patient characteristics including tumor burden. Our previous work in the immunocompetent Vk*MYC model of MM credentialed that tumor burden also correlates with resistance to a murine BCMA-BsAb, which we addressed through drug combinations with MM backbone agents. Pairing the BsAb with cyclophosphamide (Cy) was well-tolerated, reversed primary resistance, reshaped an immunosuppressive tumor microenvironment (TME) and led to durable responses with immunosurveillance in all but the most aggressive settings. Here, we aimed to improve the BCMA-BsAb and Cy combination strategy with a goal of curing models representing clinically relevant Cy-refractory aggressive settings, by adding T cell checkpoint blockade. Despite a lack of single agent activity of checkpoint inhibitors in MM (and in our models), PD1 and TIGIT are relevant targets as their expression on T cells correlates with both primary and acquired resistance to BCMA-directed cell therapies. Our team hypothesized that blockade of either PD1 or TIGIT with concurrent BCMA-BsAb and Cy would deepen anti-MM responses and provide a more durable immunotherapy regimen by sustaining activation of the T cell repertoire. Methods: We preclinically tested the ability of two weeks of concurrent dosing of BCMA-BsAb with Cy, checkpoint inhibitors targeting either PD1 or TIGIT, or the triple combination to overcome primary resistance and improve survival in a Cy-resistant transplantable Vk*MYC model of MM. We followed the response longitudinally in the blood, and in the bone marrow and spleen after complete responses were achieved at five weeks. Results: The double combination of a checkpoint inhibitor to the BCMA-BsAb was well-tolerated and abolished primary resistance, yet, failed to provide curative responses. Whereas the triple combination with either PD1 or TIGIT blockade remained safe, provided robust initial responses that overcame primary resistance and resulted in durable complete and curative responses in the entire cohort. These survival benefits represent significant improvements over control combinations tested here but also over all historical preclinical anti-MM therapy tested in our models to date. Both PD1 and TIGIT blockade with Cy/BCMA-BsAb triple combinations delivered a similar multiphasic potent anti-tumor response. This response was first characterized by highly effective T cell-mediated lysis of the tumor, as evidenced by deep reduction of M-spike after two weeks of treatment. As we previously published, the addition of Cy to BCMA-BsAb rapidly reshapes the TME by depleting Tregs and exhausted T cells and brings naive T cells to the tumor. However, we observed elevated T cell checkpoint expression at times of BsAb-relapse in aggressive models, suggesting that blockade of these pathways may help sustain T cell activation. Indeed, the ensuing T cell response in the triple combination gradually builds and encourages a pool of less terminally differentiated T cells to proliferate to allow for a more sustained response with repeat dosing. For the first time, we observe that the triple combination significantly engages the CD4 T cell compartment, which can be essential for a complete anti-tumor response. At the time of complete response, single cell analysis displays that T cells retained in the BM were less exhausted and defined by elevated CCR2 expression, a known marker of activated memory T cells, suggestive of ongoing immunosurveillance. Conclusions: Here we demonstrated through controlled in vivo experimentation how the addition of Cy reverts primary resistance to BCMA-BsAb by reshaping the TME in our Cy-insensitive preclinical model. Adding checkpoint inhibitors targeting TIGIT or PD1 further improves the durability of BsAb and Cy combination to cure all mice in these cohorts, by critically lysing the tumor, resetting the suppressive TME, and instigating a gradual, multiphasic, and sustained anti-MM T cell response. The concurrently delivered triple combination is well tolerated and provides optimal temporal control of MM especially in a preclinical model reflecting primary resistance mediated by high tumor burden and may have translational benefit to MM patients.

Introduction Poor diet quality, elevated body mass index (BMI), insulin resistance, microbiome dysbiosis, inflammation as well as immune dysfunction have all been implicated in progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to multiple myeloma (MM). Plant-based diets have been associated with reduced risk of MGUS and MM in epidemiological studies. Whether a dietary intervention can impact these modifiable risk factors and delay disease progression has not been investigated. Methods We conducted a pilot, single-arm trial providing controlled high fiber plant based dietary (HFPBD) intervention for 12 weeks and health coaching for 24 weeks in 20 patients with MGUS/SMM with BMI≥25 (NCT04920084). The primary endpoint was feasibility (adherence and BMI reduction). Secondary and exploratory endpoints included quality of life, metabolic markers (insulin, adiponectin, leptin, LDL cholesterol), microbiome (diversity and composition by 16S and shotgun sequencing) and immune profiling in peripheral blood (flow cytometry and Olink inflammation panel) and bone marrow (scRNAseq and ATACseq). We also conducted an in vivo study in transgenic Vk*MYC mice fed a high fiber or control diet at the phase of mouse(m)SMM. The mice were monitored for the progression to active mMM. Differences in microbiome and immune response profiles between high fiber- and control-treated mice were assessed via microbiome 16S sequencing and flow cytometry. Results The HFPBD intervention was safe, feasible, improved quality of life and addressed modifiable risk factors - metabolic profile (improved BMI, insulin resistance, adiponectin leptin ratio), microbiome profile (increased alpha-diversity and butyrate producers) and immune (decreased inflammation and increased anti-inflammatory classical monocyte) subsets. A reduction in long-term progression trajectory was observed in 2 patients. Consistently, the high fiber diet delayed progression from mSMM to mMM in Vk*MYC mice increasing median progression-free survival from 12 weeks in the control arm to 30 weeks in the high fiber diet intervention arm. Additionally, in the intervention arm 40% of mice did not progress to mMM during the study period whereas all mice in the control arm progressed. Both human and mouse data showed that HFPBD modulated gut microbiota composition favoring the expansion of butyrate-producing bacteria. Additionally, short-chain fatty acids were increased in the feces of mice fed a high fiber diet. Integrated analysis from human bone marrow and peripheral blood analysis indicated that the dietary intervention reduced inflammatory biomarkers and skewed the immune response towards T helper and CD14+ monocytes. Consistently, the bone marrow of Vk*MYC mice fed high fiber diet was more infiltrated by IFNg-producing T lymphocytes while displaying less exhausted T cells and immunosuppressive myeloid cells. Conclusion This is the first interventional clinical trial and in vivo study to show that a HFPBD intervention may delay progression from MGUS/SMM to MM. To our knowledge there has been no similar dietary interventional study in hematological cancers or solid tumors. Together our in vivo and clinical data support the beneficial anti-inflammatory role of a HFPBD providing a link between diet, microbiota, and immune modulation to delay disease progression in MGUS/SMM.

DISCUSSION AND CONCLUSION Chimeric antigen receptor (CAR)-T cells directed against B-cell maturation antigen (BCMA), have yielded impressive results in clinical trials for multiply relapsed/refractory multiple myeloma (MM). However, progression-free survival is short, demonstrating a need for improvements. While numerous advances have been made to improve this treatment modality, these have not always translated to superior clinical outcomes. A major limitation is that novel therapeutics are often not tested in a relevant in vivo model. These novel strategies have been largely tested only in immunodeficient models of MM. To better recapitulate the host-tumour-effector cell interactions, particularly in the TME, we sought to develop a system to study CAR-T cell activity in the Vk*MYC model of MM. Here, we report on the first known successful model of CAR-T cell activity in an immunocompetent model of MM. We sought to identify a BCMA CAR construct that is capable of binding to both human and mouse BCMA. A novel nanobody recognizing BCMA was developed by immunizing llamas with human BCMA and the resulting single-domain antibodies (sdAbs) were subsequently isolated and cloned. These sdAbs were then screened for binding to mouse BCMA. A binder capable of recognizing both human and mouse BCMA was identified and cloned into our murine CAR construct, with CD28 and CD3z signaling domains, herein referred to as sdBCMA2.CD28.CD3z. Murine CAR-T cells were then generated by transducing activated healthy murine splenocytes with a retroviral vector carrying the sdBCMA2.CD28.CD3z construct. These CAR-T cells were then characterized in vitro using standard cytotoxicity, proliferation, and cytokine release assays. To assess activity in an immunocompetent model, female C57BL/6J mice were injected intravenously with Vk12598 Vk*MYC cells. CAR-T cells were later administered intravenously. Mice were bled weekly to quantify M-protein disease burden by serum protein electrophoresis and for phenotypic characterization of circulating cells. When co-cultured with BCMA+ human MM cell lines and with Vk*MYC cell lines, murine sdBCMA2.CD28.CD3z CAR-T cells effectively lysed target cells, proliferated, and released inflammatory cytokines. To assess the in vivo activity of these CAR-T cells, we injected intravenously female C57BL/6J mice with Vk12598 cells, and then administered sdBCMA2.CD28.CD3z CAR-T cells intravenously. While there was some evidence of survival improvement, the median overall survival (mOS) of 35 days compared to 28 days, for CAR-T vs. Mock T-cells, respectively, the difference was non-significant, and the survival benefit was not durable. To address this, we incorporated lymphodepletion by total body irradiation (TBI) followed by CAR-T cell administration the next day. By incorporating lymphodepletion prior to CAR-T cell administration we were able to significantly reduce M-protein surrogates of disease burden, as well as achieve durable remissions in the TBI + CAR-T arm with an mOS not yet reached after experimental day 91, compared to an mOS of 42 days in the TBI + Mock T-cell arm. The addition of TBI also significantly improved the engraftment of CAR-T cells, as assessed by flow cytometry. The TBI + CAR-T arm also had significantly increased IL-6, IL-1b, IL-2, and TNF-a in the serum, compared to the TBI + Mock arm, at 7 days post administration of CAR-T cells. The durable remission achieved using the combination of our novel sdBCMA2.CD28.CD3z CAR-T cells with lymphodepletion is a pivotal advancement in modelling MM CAR-T cell activity in this clinically relevant model system. Our approach represents the first time that CAR-T cells were successfully used to treat the Vk12598 model of MM that faithfully recapitulates relapsed MM. The incorporation of lymphodepletion significantly improves CAR-T cell engraftment, in line with clinical CAR-T cell therapy paradigms. The cytokine profile observed is consistent with the cytokine elevations typically observed after CAR-T cell administration in patients. As our construct is capable of binding to both human and mouse BCMA, we are therefore able to fully characterize our lead assets in a relevant model system. Our strategy will enable further research to dissect host-tumour-effector cell interactions, to better inform the design and characterization of novel CAR-T cell therapies in MM.