Bone Marrow Tumor Microenvironment Research Articles

Abstract PO-028: A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma

Abstract Background. Small molecules targeting the B-cell receptor (BCR) signaling, including PI3K and BTK inhibitors, have represented a step forward for managing lymphoma patients. However, resistance often develops, negatively impacting the benefit of patients exposed to drugs. Thus, developing preclinical tools to identify modalities to overcome this phenomenon is important. The bone marrow is commonly involved in marginal zone lymphoma (MZL) patients, and it might represent a safe environment where lymphoma cells escape therapy. Bone marrow stromal cells (BMSCs) induce proliferation and drug resistance in several tumor entities, mainly through the release of cytokines. To study potential resistance mechanisms to BCR targeting agents, we aimed to develop a 3D co-culture system in which primary BMSCs were used to model the bone marrow tumor microenvironment while exposing an MZL cell line to the PI3K inhibitor copanlisib. Methods. The MZL cell line VL51 was cultured in 3D fibrin gel (10mg/mL), either alone or with primary BMSCs, in 96-well plates using the OT-2 liquid handler (Opentrons) for high-throughput seeding purposes. Copanlisib treatment started on day 3. The effects of the drug were assessed through morphological observations and quantitative measurements of images taken on days 3, 5, and 7 in the presence or absence of BMSCs. On day 7, supernatants were analyzed for 105 secreted factors using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems / Bio-Techne). Results. We observed differences in VL51 growth pattern in the different conditions. Most importantly, the co-culture with BMSCs significantly (p=0.025) decreased VL51 sensitivity to copanlisib. When not treated, VL51 cells formed significantly larger clusters (p<0.001) in the presence of BMSCs. However, upon copanlisib treatment, VL51 cells formed smaller and more distributed clusters comparable in size to those in the mono-culture. Subsequent analyses of the supernatants from the mono- or co-cultures following DMSO or copanlisib treatment identified 14 differentially secreted cytokines among the different conditions. Validation experiments were done using recombinant proteins in 2D cultures for 6 factors. Among these, recombinant IGFBP-3, serpin E1, and PTX-3 significantly decreased VL51 sensitivity to copanlisib in 2D cultures (p=0.05, 0.01, and 0.04, respectively). Validation experiments are ongoing with additional in vitro models and using BTK inhibitors. Conclusions. We created a high-throughput 3D co-culture MZL system and found that BMSCs reduce sensitivity to copanlisib in MZL models. Secretion of IGFBP-3, Serpin E1, and PTX-3 reduced response to copanlisib. This 3D-model will be used to assess resistance mechanisms to clinically relevant compounds and to evaluate alternative therapeutic approaches to improve the treatment of lymphoma patients. Citation Format: Alex Zadro, Alberto Arribas, Maria Vittoria Colombo, Eleonora Cannas, Filippo Spriano, Luciano Cascione, Chiara Arrigoni, Christian Candrian, Matteo Moretti, Francesco Bertoni. A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-028.

Iberdomide increases innate and adaptive immune cell subsets in the bone marrow of patients with relapsed/refractory multiple myeloma

Iberdomide is a potent cereblon E3 ligase modulator (CELMoD agent) with promising efficacy and safety as a monotherapy or in combination with other therapies in patients with relapsed/refractory multiple myeloma (RRMM). Using a custom mass cytometry panel designed for large-scale immunophenotyping of the bone marrow tumor microenvironment (TME), we demonstrate significant increases of effector T and natural killer (NK) cells in a cohort of 93 patients with multiple myeloma (MM) treated with iberdomide, correlating findings to disease characteristics, prior therapy, and a peripheral blood immune phenotype. Notably, changes are dose dependent, associated with objective response, and independent of prior refractoriness to MM therapies. This suggests that iberdomide broadly induces innate and adaptive immune activation in the TME, contributing to its antitumor efficacy. Our approach establishes a strategy to study treatment-induced changes in the TME of patients with MM and, more broadly, patients with cancer and establishes rational combination strategies for iberdomide with immune-enhancing therapies to treat MM.

TIM-3+ CD8 T cells with a terminally exhausted phenotype retain functional capacity in hematological malignancies.

Chronic antigen stimulation is thought to generate dysfunctional CD8 T cells. Here, we identify a CD8 T cell subset in the bone marrow tumor microenvironment that, despite an apparent terminally exhausted phenotype (TPHEX), expressed granzymes, perforin, and IFN-γ. Concurrent gene expression and DNA accessibility revealed that genes encoding these functional proteins correlated with BATF expression and motif accessibility. IFN-γ+ TPHEX effectively killed myeloma with comparable efficacy to transitory effectors, and disease progression correlated with numerical deficits in IFN-γ+ TPHEX. We also observed IFN-γ+ TPHEX within CD19-targeted chimeric antigen receptor T cells, which killed CD19+ leukemia cells. An IFN-γ+ TPHEX gene signature was recapitulated in TEX cells from human cancers, including myeloma and lymphoma. Here, we characterize a TEX subset in hematological malignancies that paradoxically retains function and is distinct from dysfunctional TEX found in chronic viral infections. Thus, IFN-γ+ TPHEX represent a potential target for immunotherapy of blood cancers.

Prognostic impact of the bone marrow tumor microenvironment, HLA-I and HLA-Ib expression in MDS and CMML progression to sAML

ABSTRACT Genetic aberrations and immune escape are fundamental in MDS and CMML initiation and progression to sAML. Therefore, quantitative and spatial immune cell organization, expression of immune checkpoints (ICP), classical human leukocyte antigen class I (HLA-I) and the non-classical HLA-Ib antigens were analyzed in 274 neoplastic and 50 non-neoplastic bone marrow (BM) biopsies using conventional and multiplex immunohistochemistry and correlated to publicly available dataset. Higher numbers of tissue infiltrating lymphocytes (TILs) were found in MDS/CMML (8.8%) compared to sAML (7.5%) and non-neoplastic BM (5.3%). Higher T cell abundance, including the CD8+ T cell subset, inversely correlated with the number of pathogenic mutations and was associated with blast BM counts, ICP expression, spatial T cell distribution and improved patients’ survival in MDS and CMML. In MDS/CMML, higher PD-1/PD-L1/PD-L2 and HLA-I, but lower HLA-G expression correlated with a significantly better patients’ outcome. Moreover, a closer spatial proximity of T cell subpopulations and their proximity to myeloid blasts showed a stronger prognostic impact when compared to TIL numbers. In sAML – the continuum of MDS and CMML – the number of TILs had no impact on prognosis, but higher CD28 and HLA-I expression correlated with a better outcome of sAML patients. This study underlines the independent prognostic value of the tumor microenvironment in MDS/CMML progression to sAML, which shows the most pronounced immune escape. Moreover, new prognostic markers, like HLA-G expression and spatial T cell distribution, were described for the first time, which might also serve as therapeutic targets.

Role of Microenvironment in Influencing the Activity of CD19 CAR T Cells in B-Cell Precursor Acute Lymphoblastic Leukemia (B-ALL)

Background: As a therapy based on immune cells, CAR T cells treatment elicits an acute response involving both innate and adaptive immunity which is followed by a process of resolution. A major interest is to identify, within the tumor microenvironment (TME), factors that influence the activity and potency of anti-CD19 CAR T cells. The B-ALL TME provides factors that protect leukemia and, conversely, suppress effector T cells. However, studies on the interaction between the bone marrow (BM) TME and CAR T cells are still limited. We hypothesized that the immunological niche of the BM reacts to CAR T-cell-mediated inflammation by modulating the immune response through the activation of inhibitory pathways and molecules. Methods: We conducted an academic phase I/II study (NCT03389035) with donor-derived anti-CD19 CAR T cells generated with Sleeping Beauty transposon (CARCIK-CD19) in B-ALL patients relapsed after alloHSCT. Hematology laboratory data, CAR T,and CD3+ cell kinetics were analyzed in 43 pediatric and adult B-ALL patients who underwent commercial and investigational anti-CD19 CAR T cell treatment. The scRNA-seq libraries of BM samples were generated using Chromium Single Cell 5′ Reagent Kit of the 10x Genomics (v3.1). Validation was performed in 20 pre-/post- CAR-T matched BM samples by a 30-color flow cytometry panel. Results: Following the peak of expansion, CAR T-cell counts decreased in the third and four-week post-infusion. A similar dynamic of expansion and contraction was observed in CD3+ T cells, total white blood cells (WBC), and monocytes, indicating that a widespread resolution of CAR T-driven inflammation occurs during the first month after infusion. Interestingly, the intensity of the response seems to differ depending on the cell product used. To elucidate the mediators involved in the resolution of CAR T-cell-mediated inflammation, the single-cell transcriptome of patients' BM cells at early time points post-CAR T-cell infusion (1-2 months) was compared to pre-treatment samples at the moment of disease relapse and the infusion products. Eighteen sequencing libraries were analyzed, 6 libraries per patient, for a total of 71407 high-quality cells at an average of 65000 reads per cell. Unbiased clustering of BM cells and infusion products was performed by UMAP embedding. Notably, extensive integration of the dataset coming from the individual patients was observed.The more representative clusters were classified into infusion product, CD4 and CD8 endogenous population, B cells, myeloid cells, pDC, NK, and NK-T cells. We observed profound changes in the composition of BM after CAR T cells infusion compared to pre-treatment samples. Complete disappearance of the B cells associated cluster was observed after treatment. CAR T-cell treatment generated a remodeling of the BM microenvironment, with an increase in the myeloid cells (specifically monocytes), NK, NK-T, and exhausted CD8+ T cell populations. After CAR T-cell infusion, myeloid cells displayed a higher resemblance to myeloid-derived suppressor cells (MDSCs). GeneSet Enrichment Analysis (GSEA) showed significant enrichment in pathways associated with immunosuppression both in the myeloid compartment and in endogenous T cells and in CAR T cells. Of note, the genes involved are also strictly correlated to the generation of terminally exhausted T cells and the emergence of MDSCs. Spectral flow cytometry-based experiments validated our observation of an increase of monocytes, MDSC-like cells, NK, NK-T, and CD8 terminal cells. Modeling intercellular communication using NicheNet suggested the induction of immunosuppressive genes in myeloid cells by both CAR T cells and endogenous T cells. Using the same tool with all niches as the sender and T cells as the receiver, we observed that myeloid cells induced significant signaling in both CAR T cells and endogenous T cells. Conclusions: Through sc-RNAseq and spectral flow cytometry, we have characterized changes in the BM TME after CAR T cell therapy. Specifically, CAR T cells-mediated myeloid activation is associated with pathways of immune dysregulation that may dampen CAR T cell expansion and antagonize the effects of the therapy. Furthermore, these data support the hypothesis that transcriptomic interrogation can infer pathways relevant to the communication between CAR T cells and the TME.

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.

Exosomal Vesicles: A Therapeutic Strategy Against Severe Sars-Cov-2 in Multiple Myeloma Patients

During the early phase of the pandemic, studies revealed that COVID-19 patients with multiple myeloma (MM) had significantly higher hospitalization rates (56%), with 20% requiring critical care and 18% facing an increased risk of mortality. MM patients undergoing anticancer therapy showed a 55% higher risk of mortality when infected with COVID-19. Even in the post-pandemic world, MM patients remain at heightened risk for breakthrough infections and severe SARS-CoV-2 due to their immunocompromised status, necessitating hospitalization. While both active and remission MM patients exhibited worse outcomes than COVID-19 patients without cancer, limited research exists to elucidate the underlying causes. In this study, we aimed to (1) develop a model elucidating the pathogenesis and interplay between MM and severe SARS-CoV-2, and (2) explain the efficacy and therapeutic potential of Exosomal Vesicles (EVs) as an intervention against severe SARS-CoV-2, supported by clinical evidence. Severe SARS-CoV-2 infection triggers multi-organ dysfunction through IL-6 mediated inflammation. Concurrently, malignancies like multiple myeloma cause apoptosis via hypoxia-induced metabolic changes, also involving IL-6. Within the tumor microenvironment, IL-6 propagates inflammation, tumorigenesis, angiogenesis, invasion, and metastasis, while inhibiting apoptosis. In MM, IL-6 serves as a survival factor, impeding apoptosis in myeloma cells and reshaping the bone marrow's tumor microenvironment, resulting in disease progression. In this model, infection with severe SARS-CoV-2 exacerbates the pro-inflammatory state in MM patients due to increased IL-6 production, stimulating increased complement and acute phase reactant production, ultimately resulting in severe systemic injury ( Figure 1). Given the virus's complexity, therapeutic options against severe SARS-CoV-2 remain limited, especially in the immunocompromised. However, EVs have shown promising therapeutic efficacy in severe SARS-CoV-2 cases, acting as both SARS-CoV-2 entry neutralizers and immunomodulators. Their capacity to deliver therapeutic cargo enhances the immune response and associated therapeutic interventions in immunocompromised individuals, while promoting angiogenesis and tissue repair. This multi-faceted therapeutic approach enables EVs to target various points in the severe SARS-CoV-2 pathway, neutralizing viral replication and mitigating systemic injury ( Figure 1). Clinically, EVs have demonstrated an impressive overall survival rate of over 90% in severe SARS-CoV-2 patients aged 18 or older, persisting for at least six months after treatment. In conclusion, EVs have proven to be safe and well-tolerated in severe COVID-19 patients, leading to improved survival rates. The unique biology of EVs allows them to effectively target disease pathogenesis at multiple steps, mitigating direct injury, suppressing IL-6, and reducing systemic inflammation, while promoting tissue repair. Moreover, their potential to work individually or synergistically with other therapeutic interventions makes them valuable in countering the virus's heterogeneity and mutational adaptations. Notably, EVs exhibit limited drug interactions and side-effects, making them a viable approach to combat SARS-CoV-2 without disrupting the treatment regimens of MM patients.

High Dimensional Profiling of Patient-Derived Multiple Myeloma Bone Marrow Specimens Treated with an FcRH5-Targeted Bispecific Ex Vivo

Background: Cevostamab is an FcRH5 (Fc receptor-homolog 5) targeted T cell-dependent bispecific (TDB) currently being evaluated in the clinic, with promising activity and favorable safety profile as a monotherapy in patients (pts) with heavily pre-treated Relapsed/Refractory multiple myeloma (MM) (NCT03275103, Trudel et al. ASH 2021). Cevostamab selectively engages and activates T cells in the proximity of MM cells in the bone marrow (BM) to drive tumor cell killing. To dissect the transcriptional and functional impact of cevostamab treatment on the MM BM tumor microenvironment (TME) and identify factors linked to enhanced killing, we established an ex vivo TDB killing assay with MM pt BMMC (BM mononuclear cells) samples followed by detailed characterization of immune and tumor cells. Methods: We tested commercially procured frozen BMMC samples from 7 pts with Stage I or Stage III Newly Diagnosed Multiple Myeloma (NDMM) with no known prior treatments (except a paired, longitudinally collected sample) and median age of 67.5 years. BMMC samples containing endogenous T and MM cells were treated ex vivo with 10 ug/mL of either a non-targeted TDB (considered as baseline) or a research grade cevostamab surrogate (FcRH5 TDB) for 16 to 48 hours (hr). T cell enumeration and MM killing (CD138 + CD38 +) was evaluated at 48 hours by flow cytometry. Ex vivo response was normalized by T cell quantity and defined as % tumor cell killing per 10 3 T cells present in the assay, which was used to classify samples as Responders (R) (>= 10%) or Suboptimal Responders (SoR) (< 10%). Single cell RNA-seq (scRNA-seq) was performed at 16, 24 and 48 hr post treatment. Tumor-specific genes enriched in SoRs at baseline were investigated for prognostic value by examining their bulk gene expression profile in baseline tumor samples (CD138-enriched) from pts (N=1150) treated with immunomodulatory drug, proteasome inhibitors, or combinations thereof (NCT01454297, MMRF CoMMpass IA22). Results: We first examined the T cell compartment by scRNA-seq profiling. Upon FcRH5 TDB treatment, clustering analysis suggested that CD4 and CD8 T cells undergo extensive transcriptional changes compared to control TDB (Fig 1A). A TNF and LTA-expressing CD4 memory cluster was enriched upon treatment in both SoRs and Rs, but to a greater extent in Rs. A similar trend was observed with an IFNG and CCL4-enriched CD8 effector-like proliferating T cell cluster. Transcriptional profiles of MM cells suggested inter- and intra-pt tumor heterogeneity at baseline. MM cells in SoRs at baseline had higher expression of genes like CD44, SRGN, CCND2 and SLAMF1. Of these genes, higher expression of CD44 or SRGN was found to be significantly associated with worse outcome to 1L treatments. Exposure to FcRH5 TDB altered tumor cells from all pts (Figure 1B), resulting in significant upregulation of genes such as HLA-E, SOCS1, and TNFSF10. Increase in CXCL9 expression upon treatment was found to be higher in Rs than SoRs. Discussion: Our work provides a novel ex vivo approach for unbiased, high dimensional pharmacodynamic profiling of functional and transcriptomic changes in pt samples by combining TDB treatment with scRNA-seq. Consistent with the expected mechanism of action and preliminary biomarker analysis reported for cevostamab (Nakamura et al. ASH 2020), FcRH5 TDB led to increased expression of genes in T cells associated with infiltration, activation and effector function. It also induced transcriptional changes in MM cells regardless of the ex vivo response status, including upregulation of genes associated with T and NK cell infiltration and antigen presentation. For certain genes, a greater magnitude of these changes was observed in Rs. These identified genes, and enriched clusters will be examined in clinical RNAseq data from cevostamab-treated pts. It remains to be determined whether our results reflect treatment effects that are applicable to other targeted bispecifics in MM. Overall, our assay setup can serve as a platform to generate novel hypotheses, corroborate clinical observations, identify pt-specific features driving improved or dampened response, and test novel combinations for improved killing. Figure 1 : UMAP (Uniform Manifold Approximation and Projection) plot of all (A) T cells and (B) tumor cells shows distinct clustering of cells by treatment type for all pts (red: FcRH5 TDB; blue: non-targeted TDB)

Self-Supporting Human Acute Myeloid Leukemia (AML) Bone Marrow Organoids Established from Patient-Derived Xenografts Mimic the Human AML Microenvironment, with Distinct Cytokine Kinetics Correlating with Dynamic Cell Proliferation and Metabolic Shifts

Hallmarks of acute myeloid leukemia (AML) include (1) formation of a self-supporting and self-regulating tumor microenvironment (TME) in the bone marrow (BM) rendering pathological niches that orchestrate retention, proliferation and survival of AML blasts, and (2) a dynamic metabolism that promotes therapy resistance and disease progression. Efforts to understand AML biology and enhance current therapies are hampered by the paucity of state-of-the-art human models that can recreate the three-dimensional (3D) BM TME and recapitulate the heterogeneity, complexity, dynamics, and metabolic shifts observed in human disease. Previously, we described the development of the first long-term self-propagating serum- and cytokine-free human BM organoid generated from an AML patient-derived xenograft (PDX) that captured metabolic shifts during AML progression. Here, we characterize the dynamic changes observed in the self-constituted TME with respect to AML proliferation, metabolism, and upon reconstitution of secondary organoids. BM was collected from NSG mice engrafted with human primary AML cells (PDX-AML model) or naïve control mice and seeded into polyurethane scaffolds in serum- and cytokine-free medium at 4e6 cells per scaffold, optimized previously. Scaffolds were recharged with autologous PDX-AML BM on day (D) 13 and cultured until D70. Organoids were assessed for (1) inter- and intra-scaffold AML kinetics, (2) the TME, (3) metabolic composition and (4) propagation into secondary organoids. AML cell kinetics from organoid culture supernatants reflected those of scaffold-extracted populations (n=3). CD33 +CD44 + AML cells expanded and/or were maintained from 7% (D0) to >10% (D70), being >15% at most culture timepoints. Confocal microscopy revealed reconstitution of typical AML-TME niche interactions within organoids, including expression of fibronectin, VCAM-1 and N-Cadherin, and formation of proliferative niches composed of AML cells with heterogeneous Osteopontin- and Osterix-expressing support cells. Human AML-supportive cytokines were consistently observed in organoid supernatants (N=3), with SDF-1, IL-1α, IL-8, GM-CSF, FGF-2 and Osteopontin present throughout the culture; some cytokines had biphasic kinetics with factors critical for TME formation present early (e.g. Osteopontin, GM-CSF), and those required for AML proliferation dominant later (e.g. FLT3L, IL-8), coinciding also with metabolic shifts during culture. Despite the absence of IL-6, single molecule RNA fluorescence in situ hybridization confirmed IL-6 expression by niche-resident AML blasts suggesting autocrine signaling networks beyond those identified in supernatants. Cells from D70 organoids were able to re-establish secondary organoids (n=2) which also exhibited long-term (D70) AML maintenance (>11% blasts), and regenerated the AML TME. Previously, we identified 3 culture stages defined by metabolic shifts: 1) pre/early recharge (D4-D19), (2) intermediate (D25-D40) and, (3) late (from D55) where organoids exhibited highest proliferation. To further characterize these shifts, intracellular metabolites were extracted from organoids (n=5) at D10, D25, D40, D55 and D70, analyzed with gas-chromatography mass spectrometry (GC-MS) and evaluated through unsupervised hierarchical clustering and multivariate analysis. Significance Analysis of Microarrays (SAM) analysis demonstrated dysregulation of alanine, serine, isoleucine, glycolate, hexanoic acid and glycolysis when comparing stage 1 with stage 2, and in isoleucine, leucine, serine, succinate and tryptophan metabolism when comparing stage 2 with stage 3. When proliferative and non-proliferative timepoints were compared, amino acid and glycolysis-driven metabolic switches were identified, typical of AML metabolism. We have characterized a long-term 3D human AML BM biomimicry in serum- and cytokine-free conditions with a self-organizing TME that recapitulates many features of AML BM, including robust human AML cell maintenance within niches, cytokine and metabolic kinetics to support different phases of progression, and the ability to propagate into secondary organoids. This dynamic, physiologically-relevant ex vivo platform can enable studies in human AML pathogenesis/progression and is poised to test novel therapeutics directed towards AML cells, the TME and metabolic targets.

Downstream process and evaluation of the concomitant impact of a recombinant glycosylated L-asparaginase on leukemic cancer cells and the bone marrow tumor microenvironment

L-asparaginase (L-ASNase) is a life-saving medication used in the treatment of Acute Lymphoblastic Leukemia (ALL) which affects over 60,000 people yearly. However, L-ASNase still requires improvement since up to 60% of the patients develop hypersensitivity due to its immunogenicity. To address this issue, this work describes the downstream process of an L-ASNase from Erwinia chrysanthemi expressed extracellularly by Pichia pastoris with human-like glycosylation pattern and its further impact on leukemic cells co-cultured with bone marrow (BM) cytoprotective stromal cells. After size exclusion chromathography, a final yield of 54.93% was achieved and the proteomics analyses confirmed the attainment of an extremely pure enzyme. Glycosylated L-ASNase induced the complete inhibition of the proliferation of ALL and Acute Myeloid Leukemia (AML) cell lines tested, independently of the presence of BM stromal cells. However, the cytotoxic efficacy (induction of apoptosis) of glycosylated L-ASNase varied across cell lines, with ALL cell lines showing the most sensitivity. Additionally, the pro-apoptotic effect of glycosylated L-ASNase was partially inhibited by BM stromal cells. Taken together, our data warrant further investigations for the use of glycosylated L-ASNase against ALL and AML that should take into consideration the mechanisms of resistance mediated by the BM stroma for improved efficacy.

Abstract LB188: Discovery and development of fully human TACI/BCMA bispecific armored CAR for the treatment of relapsed/refractory multiple myeloma

Abstract Background: The introduction of novel targeted therapies and immunotherapies has dramatically improved responses and overall survival in multiple myeloma (MM) patients. However, greater than 50% MM achieve refractory or relapse (r/r) within five-year due to MM target heterogeneity and bone marrow tumor microenvironment (TME). The approved B-cell maturation antigen (BCMA) -directed CAR T therapies have shown unprecedented efficacy in advanced r/r MM, yet relapse remains to occur. The shedded soluble BCMA, target escape and bone marrow TME may contribute to the r/r MM mechanism through compromising clinical efficacy and durability. TACI and BCMA are co-expressed on the tumors of ~78% MM patients, and therefore, dual targeted TACI/BCMA CAR therapy could be an attractive strategy in addressing challenges of BCMA mono-CAR therapy. We have developed EPC-004, a fully human anti-TACI and BCMA bispecific armored tandem CAR, aiming to overcome BCMA target escape mechanism, reduce immunosuppression within the bone marrow tumor microenvironment, and be more persistent with engineered armor and the improved cell manufacturing process. Methods: Fully human anti-TACI, anti-BCMA and anti-PDL1 scFv antibodies with a broad affinity range and epitope coverage have been discovered by proprietary mRNADisTM mRNA display and live cell selection platform. The T cell modulatory activity fine-tuned IL2 was discovered by the mSCAFoldTM technology through the structure guided mutagenic library design and high throughput systemic screening. The anti-PDL1 scFv was then fused to the engineered IL2 to generate an armor molecule to enhance CAR activities and persistence. Finally, EPC-004, an optimal bispecific tandem TACI/BCMA armored CAR candidate, was developed by precision engineering of multiple pairs of anti-TACI and BCMA scFvs with monospecific and bispecific target binding properties, spacers and scFv orientation. Results: EPC-004 demonstrated both mono-specific and bispecific TACI and BCMA target cell engagement and effective cytotoxic cancer cell killing activities. EPC-004 showed potent anti-tumor activity in MM1R, BCMA knock out or TACI knock out MM1R -luciferase orthotopic animal models, which validated the mechanism of EPC-004 in overcoming BCMA or TACI target escape resistance. Furthermore, EPC-004 showed strong persistent CAR-T activity against tumor cells in re-challenging models in mice. Importantly, the secreted armor molecule selectively activates central memory T cells and contributed to the enhanced anti-tumor activity and persistence in vivo. Conclusion: EPC-004, engineered with multi-modules and multi-mechanisms is a promising candidate which enhances anti-tumor activity, prevents target escape, reduces immunosuppression within the bone marrow tumor microenvironment. These novel mechanisms could potentially translate EPC-004 into durable clinical efficacy in treating R/R multiple myeloma. Citation Format: Garima Agrahari, Wenle Liang, Katie O'Callaghan, Keming Zhang, Ning Jiang, Xiaoyi Jin, Ryan Feng, Jenna Nguyen, Sam Hassan, Annie Luu, Tony Ruan, Kehao Zhao, Yan Chen. Discovery and development of fully human TACI/BCMA bispecific armored CAR for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB188.

Potential Multiple Myeloma Therapeutic Strategies through Targeting Macrophages and Mesenchymal Stromal Cells

Multiple Myeloma (MM), a bone marrow plasma cell hematopoietic cancer, remains a critical but incurable hematological malignancy, prone to deadly relapses even after existing treatment. In this review, I describe the origins of pro-tumor myeloma-associated macrophages. I specifically outline how classically activated anti-tumor macrophages that home to the cancerous bone marrow tumor microenvironment is polarized into pro-tumor macrophages. We then explain how these myeloma-associated macrophages play an important role in supporting multiple myeloma by enabling drug resistance, improved growth, angiogenesis, and protection. We also describe several treatments in development aimed to sever the supportive link between myeloma-associated macrophages and MM by blocking signaling pathways, destroying, or repolarizing macrophages and even preventing macrophage polarization in the first place. We conclude that careful study is needed to improve the reliability of targeting myeloma-associated macrophages to reduce MM relapse and comprehensively treat this cancer.

TIGIT inhibition and lenalidomide synergistically promote antimyeloma immune responses after stem cell transplantation in mice.

Autologous stem cell transplantation (ASCT) with subsequent lenalidomide maintenance is standard consolidation therapy for multiple myeloma, and a subset of patients achieve durable progression-free survival that is suggestive of long-term immune control. Nonetheless, most patients ultimately relapse, suggesting immune escape. TIGIT appears to be a potent inhibitor of myeloma-specific immunity and represents a promising new checkpoint target. Here we demonstrate high expression of TIGIT on activated CD8+ T cells in mobilized peripheral blood stem cell grafts from patients with myeloma. To guide clinical application of TIGIT inhibition, we evaluated identical anti-TIGIT antibodies that do or do not engage FcγR and demonstrated that anti-TIGIT activity is dependent on FcγR binding. We subsequently used CRBN mice to investigate the efficacy of anti-TIGIT in combination with lenalidomide maintenance after transplantation. Notably, the combination of anti-TIGIT with lenalidomide provided synergistic, CD8+ T cell-dependent, antimyeloma efficacy. Analysis of bone marrow (BM) CD8+ T cells demonstrated that combination therapy suppressed T cell exhaustion, enhanced effector function, and expanded central memory subsets. Importantly, these immune phenotypes were specific to the BM tumor microenvironment. Collectively, these data provide a logical rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent myeloma progression after ASCT.

HCK Inhibition Displays Antineoplastic Activity on Acute Leukemia and Promotes Immune Regulation of the Bone Marrow Tumor Microenvironment

Background. The hematopoietic cell kinase (HCK), a Src tyrosine kinase family member, is mainly expressed in myeloid cells and lymphocytes and regulates oncogenic signal transduction pathways acting on pivotal biological processes. Increased activity of HCK, a common feature in several hematological neoplasms, modulates immune cell recruitment and activity in the tumor microenvironment (TME). HCK is also associated with the development of acute leukemia and correlates with poor prognosis, particularly, in acute myeloid leukemia (AML) patients relapsed from standard chemotherapy. In light of the urgent need for advancing targeted therapeutic options for acute leukemia, we investigated the effects of a novel HCK inhibitor. Aims. To analyze HCK gene and protein expression in leukemia patients and their impact on clinical outcomes, and to investigate preclinical effects of a novel HCK inhibitor compound, an iHCK-37 subproduct (Dr Maurizio Botta, Siena University). Methods. Analysis of HCK gene expression was performed in mononuclear cells isolated from 163 leukemia patients (median age 57.5 years [range: 18-88]) (TCGA study). HCK protein levels were analyzed in 25 bone marrow (BM) aspirates from healthy donors (HD) and untreated patients with Myelodysplastic syndrome (MDS), AML with MDS-related changes (AML-MRC), de novo AML, and acute lymphoblastic leukemia (ALL) by flow cytometry and immunofluorescence. To further explore the clinical potential of the HCK inhibitor, we conducted functional assays in 9 leukemia cell lines: AML (OCI-AML3, MOLM13, U937, KG1a), ALL (RS4;11, Jurkat), chronic myeloid leukemia (K562), and erythroleukemia (Hel). We also tested the HCK inhibitor action on 12 BM hematopoietic stem cells isolated from HD, MDS, AML-MRC and AML to verify their colony-forming potential and to investigate the mechanisms in the TME using a 3D-coculture system containing mesenchymal cells and 2D-coculture with a monocyte monolayer. In vivo, we developed a xenograft mouse model in which leukemia cells were inoculated into NOD/SCID mice flank to form tumors. Statistical analyzes were performed using Kruskal-Wallis, ANOVA or Mann-Whitney tests (p<.05). Results. HCK gene expression was increased in cells from M4 and M5 AML patients compared to patients with other AML (p<.0001). Additionally, HCK protein levels were higher in BM cells from MDS, AML-MRC, AML and ALL compared to HD. The HCK inhibitor exhibits a potent cell growth inhibition with doses ranging from 0.3 to 9.8μM in leukemia cells (KG1a: 2.0μM, HL60: 0.5μM, U937: 0.3μM, OCI-AML3: 4.2μM, MOLM13: 8.5μM, Jurkat: 3.9μM; RS4;11: 2.1μM; K562: 9.8μM; Hel: 5.0μM). Preclinical in vitro experiments demonstrated that treatment with the HCK inhibitor for 48h was sufficient to drive acute leukemia cells into irreversible cell growth inhibition and death (p<.0001). The ability of BM hematopoietic progenitors to proliferate and differentiate into colonies was significantly suppressed by the HCK inhibitor in MDS, AML-MRC and AML (p<.001) but not in HD. The HCK inhibitor led to increased apoptosis in a 3D-system of leukemia cell lines or AML patients’ cells, but had no effect on HD cells. In a 2D-coculture, programmed monocytes potentialized leukemia cell apoptosis (p<.001). In vivo xenograft studies showed a significant reduction of tumor growth (U937: 62%; OCI-AML3: 66%) and volume (U937: 68%; OCI-AML3: 71%) in HCK inhibitor-treated mice compared to the vehicle-treated group. In addition, both in vitro and in vivo protein analyses showed an abrogation of PI3K and MAPK signaling activation by a downregulation on AKT and ERK phosphorylation levels after the HCK inhibitor treatment. Conclusion. The HCK inhibitor has an antineoplastic activity onto leukemia cells without altering normal cell survival, demonstrating on-target malignant cell killing action probably through reducing the activation of the oncogenic pathways PI3K and MAPK. Interestingly, nowadays the inefficacy of standard chemotherapy is related to non-specificity between leukemic and normal hematopoietic cells and to the advantages of neoplasm cell survival due to TME protection. In this scenario, our results altogether demonstrate the efficacy of HCK inhibition and identify this compound as a promising therapeutic opportunity that could enhance the ability of reversing chemotherapy resistance. Funding: FAPESP grants #2017/21801-2, #2019/25247-5, #2021/05320-0.

Bone Marrow Endothelial Cells Increase Prostate Cancer Cell Apoptosis in 3D Triculture Model of Reactive Stroma.

Simple SummaryProstate cancer (PCa) metastasizes preferentially to the bone marrow where it becomes difficult to treat. PCa cells in the bone marrow may survive, dormant and undetected for many years before patients eventually relapse with metastatic disease. Bone marrow is a complex tissue that initially is hostile to the PCa cells, Understanding how cancer cells survive in the bone marrow and what changes to the bone microenvironment permit them to switch to an actively growing state could offer new therapeutic strategies to combat metastatic PCa. In this study, we describe a method to culture PCa cells with two other cell types from the bone marrow, stromal cells and endothelial cells, as a way to study the interactions among these cell types. We found that factors produced by bone marrow endothelial cells, but not endothelial cells from other tissues, trigger PCa cells to either die or enter a dormant state, similar to what has been observed in patients when PCa cells initially colonize the bone marrow. Further analysis of the cell interactions within the culture model described in this study will offer increased understanding of PCa interaction with the bone marrow environment.The bone marrow tumor microenvironment (BMTE) is a complex network of cells, extracellular matrix, and sequestered signaling factors that initially act as a hostile environment for disseminating tumor cells (DTCs) from the cancerous prostate. Three-dimensional (3D) culture systems offer an opportunity to better model these complex interactions in reactive stroma, providing contextual behaviors for cancer cells, stromal cells, and endothelial cells. Using a new system designed for the triculture of osteoblastic prostate cancer (PCa) cells, stromal cells, and microvascular endothelial cells, we uncovered a context-specific pro-apoptotic effect of endothelial cells of the bone marrow different from those derived from the lung or dermis. The paracrine nature of this effect was demonstrated by observations that conditioned medium from bone marrow endothelial cells, but not from dermal or lung endothelial cells, led to PCa cell death in microtumors grown in 3D BMTE-simulating hydrogels. Analysis of the phosphoproteome by reverse phase protein analysis (RPPA) of PCa cells treated with conditioned media from different endothelial cells identified the differential regulation of pathways involved in proliferation, cell cycle regulation, and apoptosis. The findings from the RPPA were validated by western blotting for representative signaling factors identified, including forkhead box M1 (FOXM1; proliferation factor), pRb (cell cycle regulator), and Smac/DIABLO (pro-apoptosis) among treatment conditions. The 3D model presented here thus presents an accurate model to study the influence of the reactive BMTE, including stromal and endothelial cells, on the adaptive behaviors of cancer cells modeling DTCs at sites of bone metastasis. These findings in 3D culture systems can lead to a better understanding of the real-time interactions among cells present in reactive stroma than is possible using animal models.

Combination therapy for treating drug-resistant multiple myeloma

Abstract Proteasome inhibitors (PIs) have remarkably improved the survival of multiple myeloma (MM) patients but dose-limiting toxicities and the development of drug resistance limit their long-term utility. Elevated TGF-β levels in MM patient sera correlate with chemoresistance, disease progression, metastasis and poor prognosis. Therefore, we evaluated the anti-MM therapeutic potential of a TGF-β type I receptor kinase inhibitor, Vactosertib. In vitro treatment of Vactosertib synergistically inhibited the growth of PI-resistant MM cells in combination with the ixazomib by suppressing TGF-β activation of Smad2/3 and expression of PSMB5, which encodes the proteasome 5 catalytic subunit targeted by PIs. Oral administration of Vactosertib as a single agent inhibited MM progression and induced a decrease in mortality and an increase in body weight of the mice bearing MM. Vactosertib alone also attenuated TGF-β activation of Smad2/3, reduced the expansion of CD11b+Gr-1+ myeloid derived suppressor cells (MDSCs) in bone marrow tumor microenvironment, and diminished the population of Foxp3+ regulatory T cells (Treg) in the spleen. Combination therapy of Vactosertib plus ixazomib was exhibited a synergistic anti-tumor effect when compared to either Vactosertib or ixazomib alone by greatly prolonged survival and significant a reduction in both MDSCs and Tregs. Taken together, our data provide the rationale for clinical evaluation of Vactosertib in MM and demonstrate proof-of-concept that combination of Vactosertib and PIs may overcome chemoresistance and enhance durable patient responses. Supported by 1R03CA259901-01A1

Natural killer cells activity against multiple myeloma cells is modulated by osteoblast-induced IL-6 and IL-10 production

BackgroundNatural killer (NK) cells are part of the innate arm of the immune system; as such NK cells can be activated rapidly to target virus-infected cells and tumor cells without prior sensitization. The human NK-92MI cell line is among the most widely used NK cell in preclinical research studies and has also been approved for clinical applications. Previous studies have shown that osteoblasts (OSB) confer drug resistance in multiple myeloma (MM) and other cancers that metastasize to the bone marrow. AimWe evaluated here how OSB, which are bone forming cells and a key cellular component of the bone marrow microenvironment, modulate the cytotoxic activity of NK-92MI cells against the MM.1S multiple myeloma cell line. MethodsThe osteoblastic niche was recapitulated with either the osteoblastic cell line hFOB 1.19 (hFOB) or primary osteoblasts (P-OSB) derived from surgical resections. Time-lapse imaging was utilized to quantify changes in MM.1S cell viability under different conditions, including: (1) Co-culture of MM.1S with NK92MI cells, (2) triple-culture of hFOB or P-OSB with MM.1S and NK-92MI, and (3) MM.1S or NK-92MI cells primed with OSB-derived supernatant. Cytokine analysis was conducted to quantify potential secreted factors associated with the protective effects of OSB. ResultsThe physical presence of OSB hindered the activity of NK-92MI cells, resulting in the increased viability of MM.1S compared to co-cultures which lacked OSB. This observation was accompanied by reduced perforin and granzyme A secretion from NK-92MI cells. Contact of OSB and NK-92MI cells also induced interleukin 6 (IL-6) and interleukin 10 (IL-10) production; two cytokines which are known to impair the NK cell immunity against MM and other cancers. OSB supernatant also conferred cytoprotection to MM.1S, suggesting a dual mechanism by which OSB may modulate both NK and MM cells. ConclusionsWe demonstrated here that OSB can negatively impact the activity of NK cells against MM. As NK cells and their chimeric antigen receptor-modified versions become more widely used in the clinic, our results suggest that understanding the role of OSB as potential immunoregulators of the NK cell-mediated cytotoxic response in the bone marrow tumor microenvironment may provide new opportunities for enhancing the effectiveness of this potent immunotherapeutic approach.

Large-Scale Mass Cytometry Reveals Significant Activation of Innate and Adaptive Immunity in Bone Marrow Tumor Microenvironment of Iberdomide-Treated Myeloma Patients

Background: Iberdomide (IBER) is a potent cereblon E3 ligase modulator (CELMoD agent) with direct anti-tumor and immunomodulatory activities in multiple myeloma (MM). An ongoing phase 1b/2a multicenter, open-label, dose-escalation study has reported favorable efficacy and safety of IBER plus low-dose dexamethasone (DEX) in heavily pretreated patients with relapsed/refractory MM (RRMM). While previous studies focused on IMiD/CELMoD agent immune effects in peripheral blood (Amatangelo, ASH 2019), a large-scale characterization of pharmacodynamics in the bone marrow (BM) tumor microenvironment (TME) has not been reported. Here, we present data on BM aspirates (BMAs) of 99 individuals pre-/post-treatment with IBER in the largest study of TME immune dynamics of RRMM patients to date.Methods: A mass cytometry (CyTOF) panel was designed/validated to capture deep and comprehensive immunophenotyping of T, B, and NK cell subpopulations. In all, staining with 37 metal-tagged Abs characterized 110 supervised cell phenotypes. Paired longitudinal assessment was conducted for viably preserved BMAs of IBER±DEX treated patients in dose-escalation of the CC220-MM-001 Ph 1b/2a study pre (SCREEN) and post treatment (cycle 2 day 15, C2D15). For a subset of patients (n=12), samples were available at disease progression (PD) . A training set analysis (n=64, data reported here) collected in North America, was validated against an independent test set (n=35) recruited in Europe. An R-based computational workflow and manual hierarchical gating identified subpopulations of B, T, NK and myeloid cells. In total, more than 5M immune cells were captured (approx. 40K per specimen). Cell populations are expressed as percentage of non-tumor bone marrow mononuclear cells (BMMC, i.e. CD45+CD66b-). P values are by Mann-Whitney U test for unpaired and Wilcoxon test for paired analyses.Results: IBER treatment resulted in profound immune shifts in the TME. B cells decreased (median SCREEN 3.3% vs C2D15 0.7%, p=0.001), with significant reduction of naïve (p=0.001) and regulatory B cells (p<0.001). Decrease occurred in CD4+ T cells (median 15.3% vs 8.9%, p<0.001), whereas CD8+ T cells increased (median 14.9% vs 17.4%). T cell subtypes showed a shift towards cytotoxic effector-memory phenotype (CCR7-CD45RA) (median 67.6% vs 80.9% of CD4+ T cells, p<0.001 and median 78.2% vs 89.2% of CD8+ T cells, p<0.001) with concurrent reduction of naive (CD45RA+CCR7+), central-memory (CD45RA-CCR7+) and EMRA (CD45RA+CCR7-) T cells. The shift towards a cytotoxic TME is confirmed by significant increases of GZMB+, HLA-DR+, ICOS+, Ki-67+, CXCR3+ and CD38+ activated CD8+ T cells (p≤0.001, paired). Conversely, CD8+ T cells expressing inhibitory checkpoints TIGIT and KLRG1 decreased significantly (p≤0.001, paired). Similar changes (with minor deviations) were noted in the CD4+ T cell compartment.NK cells increased (median 4.2% vs 8.7%, p=0.003), with increase of both CD56hi cytokine-producing (median 1.9% vs 4.5%, p<0.001) and CD16+ cytolytic (median 1.8% vs 2.6%, p=0.06) subsets. NKG2A+ and NKG2D+ subsets were increased (p<0.001, paired), as well as subsets expressing markers of activation: GZMB, ICOS, CD38 and PD-1 (p<0.05, paired). TIGIT+ NK cells decreased significantly. NKT cells were also significantly increased (median 1.4% vs 2.2%, p<0.001). Increase of NK and NKT cells expressing the activating receptor NKG2D was limited to patients achieving best response of PR or better. Additional analyses correlating immune phenotypes at baseline/post-treatment with outcomes are ongoing and will be reported at the meeting, as will data on how prior treatment (e.g. with CD38-mAb) shapes the TME at baseline and affects response.Conclusion: Our analysis on a large cohort of RRMM patients provides unique insights into the heterogeneity of the immune TME of heavily pretreated MM patients and how it changes upon treatment with IBER±DEX. We found significant increases of effector T and NK cells in paired analysis, demonstrating innate and adaptive immune enhancement in the MM bone marrow niche as an important mechanism of action. Importantly, these changes were observed throughout dose escalation and in patients previously refractory to lenalidomide/pomalidomide. The presented platform of large-scale immune profiling demonstrates a strategy to design and study rational combinations with (other) immune-enhancing therapies in MM. [Display omitted] DisclosuresAmatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Gooding: Bristol Myers Squibb: Research Funding. Jagannath: Legend Biotech: Consultancy; Bristol Myers Squibb: Consultancy; Karyopharm Therapeutics: Consultancy; Janssen Pharmaceuticals: Consultancy; Takeda: Consultancy; Sanofi: Consultancy. Pierceall: BMS: Current Employment, Current equity holder in publicly-traded company. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

The IL-2/IL-15 Mimetic NL-201 Prevents Myeloma Relapse after ASCT By Expanding Highly Cytolytic T Cells in the Bone Marrow That Are Resistant to Exhaustion

Multiple myeloma (MM) is a bone marrow (BM) resident hematological malignancy that is becoming increasingly recognized as one amenable to immunotherapy, although no therapies have yet provided durable, long-term disease control. Autologous stem cell transplantation (ASCT), the standard of care in eligible patients, provides a window for intervention with immunotherapy due to the induction of inflammation in the context of lymphodepletion at a time where there is also minimal residual disease and a disrupted tumor microenvironment (TME). We have previously established that the addition of T cells to BM grafts results in enhanced long-term myeloma control post-transplant in mice. Novel approaches aimed at improving and/or expanding the endogenous T cell response early post-ASCT may therefore prove highly effective with the benefit of avoiding ex vivo processing associated with other cellular therapies. To explore this, we utilized the IL-2/IL-15 mimetic NL-201: a de novo cytokine mimetic that signals via the beta and gamma subunits of the IL-2 receptor without engaging IL-2Rα (CD25). In pre-clinical studies, NL-201 has demonstrated the ability to signal to effector CD4 and CD8 T cells while avoiding the toxicity usually associated with IL-2 signaling via IL-2Rα.We hypothesized that NL-201 would enhance control of myeloma progression by stimulating T cell proliferation and activation early post-ASCT. We transplanted lethally irradiated Vk*MYC myeloma-bearing B6 recipients with BM and T cells graft from B6 donors and administered NL-201 from D+7 to week 6 (225 μg/kg weekly I.P). NL-201 promoted potent anti-myeloma immunity that was dependent on CD4 and CD8 T cells, but not NK cells (median survival was 68 days for control mice, unreached at >120 days for NL-201 alone or with NK depletion, 86 days for NL-201 with CD8 depletion, and 74 days with CD4 depletion; PBS vs NL-201 p<0.01; PBS vs NL-201 + αNK1.1 p<0.01; NL-201 vs NL-201 + αCD4 or αCD8 p<0.05). To further elucidate potential mechanisms of action we harvested BM from PBS and NL-201-treated mice 2 days after the last dose was administered and performed comprehensive immunophenotyping with high parameter flow cytometry. We grouped recipients based on whether they had controlled myeloma (MM-controlled) or had active disease progression at the time of harvest (MM-relapsed) to reveal immunological phenotypes that were dependent and independent of myeloma in the TME. In these experiments, all NL-201-treated recipients had controlled myeloma at time of harvest. Mechanistically, NL-201 significantly expanded the total number of CD8 T cells in the BM compared to PBS-treated mice with controlled or relapsed MM (PBS-treated mean CD8 T cell number was 1.0 x 10 5/femur vs 7.7 x 10 5/femur in NL-201-treated mice) but did not impact CD8 T cell number in peripheral blood. Memory CD8 T cells (CD44+CD62L+) were preferentially expanded, while the frequency of exhausted CD8 T cells (TOX +PD-1 +TIGIT +CD39 +; T EX) was reduced in NL-201-treated mice compared to both PBS-treated MM-relapsed and MM-controlled mice (75% T EX in PBS MM-relapsed, 15% PBS MM-controlled, 2% in NL-201; p<0.001). Surprisingly, >80% of the memory CD8 T cells in NL-201-treated mice produced granzyme B compared to <10% in PBS-treated mice. Granzyme B production was also observed in conventional CD4 T cells in response to NL-201 treatment, and the frequency of regulatory T cells was reduced by 50% after NL-201 compared to PBS MM-controlled and MM-relapsed mice (p<0.001).NL-201 expanded bone marrow resident cytotoxic memory CD8 and CD4 T cells that are resistant to exhaustion, whilst reducing the frequency of regulatory T cells in the BM TME. Together, these data highlight the promising therapeutic potential of NL-201 in multiple myeloma and support testing NL-201 in clinical trials for the treatment of hematological malignancies. DisclosuresHill: NapaJen Pharma: Consultancy; Roche: Research Funding; Syndax Pharmaceuticals: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Applied Molecular Transport: Research Funding; Compass Therapeutics: Research Funding; NeoLeukin Therapeutics: Consultancy; Generon Corporation: Consultancy.

The Combination of Anti-Tigit and Lenalidomide Promotes Synergistic Myeloma-Specific Immunity after ASCT

Multiple myeloma is a largely incurable bone marrow (BM) resident plasma cell malignancy that is increasing in incidence. Autologous stem cell transplantation (ASCT) is the current standard consolidation therapy and a subset of patients achieve durable progression free survival that is suggestive of long-term immune control. Utilizing novel preclinical models, we have provided definitive evidence that this is largely mediated by T cell-dependent myeloma-specific immunity. In both patients and preclinical models, myeloma progression is associated with T cell dysfunction and expression of multiple inhibitory receptors suggesting a loss of immunosurveillance. In mice, we have demonstrated potent anti-myeloma efficacy of TIGIT blockade in both ASCT and non-transplant settings. Here we utilized identical TIGIT Abs that do or do not Fc bind to demonstrate that immunological efficacy after ASCT was absolutely dependent on ADCC (median survival was unreached (>110 days) in Fc-binding vs 73 days in Fc-dead and 71 days in control Ig (cIg)-treated mice). Since TIGIT inhibition does not protect against myeloma relapse in all mice, it is apparent that combinational approaches are required to target non-responders. Therefore, we hypothesized that TIGIT blockade could be combined with immunomodulatory drugs (IMiDs) to provide synergistic anti-myeloma activity after ASCT.To that end, we utilized CRBN transgenic mice to investigate the efficacy of TIGIT blockade in combination with lenalidomide, the standard of care IMiD used in maintenance therapy after clinical ASCT. Briefly, B6 Vk*MYC myeloma-bearing (MM-bearing) mice were lethally irradiated and transplanted with B6 bone marrow (BM) and a suboptimal dose of T cells followed by anti-TIGIT or control Ig (100 mg twice weekly) for 5 weeks with lenalidomide (50 mg/kg daily gavage) or control diluent from D+14 for 3 weeks (Figure 1A). The combination of anti-TIGIT and lenalidomide provided synergistic anti-myeloma efficacy evidenced by prolonged median survival (109 days in combination vs < 60 days in monotherapy/control-treated mice, p<0.01; Figure B). Myeloma M bands were also suppressed in the combination treated mice relative to monotherapy or cIg-treated mice (p<0.01; Figure 1). Analysis of BM CD8 T cells 6 weeks after ASCT demonstrated that combination therapy significantly decreased terminal exhaustion (TOX + TIM3 + CD101 + PD-1 + DNAM-1 ─) with an average of only 20% of CD8 T cells with an exhausted phenotype in the combination group compared to greater than 50% exhausted CD8 T cells in monotherapy or cIg-treated mice (p<0.05; Figure 1C-D). The combination also increased the frequency of central memory and tissue-resident memory subsets (CD49b +CD69 +; p<0.05; Figure 1C-D), and increased IFNγ production from activated (PD-1 +; p<0.05; Figure 1E) cells compared to monotherapy or control Ig-treated mice. Importantly, these phenotypic changes were specific to the BM tumor microenvironment as we observed no effect of combination or monotherapy treatment on CD8 or CD4 T cells in peripheral blood.In sum, these data provide a strong rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent the progression of myeloma. [Display omitted] DisclosuresDriessens: iTeos Therapeutics: Current Employment, Current equity holder in publicly-traded company. Holmberg: Up-To-Date: Patents & Royalties; Bristol Myers Squibb: Research Funding; Janssen: Research Funding; Merck: Research Funding; Millennium-Takeda: Research Funding; Sanofi: Research Funding; Seattle Genetics: Research Funding. Hill: NeoLeukin Therapeutics: Consultancy; Compass Therapeutics: Research Funding; NapaJen Pharma: Consultancy; Generon Corporation: Consultancy; Roche: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Applied Molecular Transport: Research Funding.