Multiple myeloma (MM) is an incurable hematologic cancer where malignant plasma cells accumulate in the bone marrow (BM), with disease progression highly dependent on cellular interactions. Obesity is a major risk factor for myeloma, however the underlying mechanisms are unclear. Here we used a murine myeloma model to show that a high cholesterol diet increases local and circulating levels of low density lipoprotein cholesterol (LDL), increasing bone marrow tumour burden in vivo. Exogenous LDL induced bortezomib-specific drug resistance in metabolically stressed myeloma cells in vitro and ex vivo. RNA-seq analysis revealed bortezomib-induced changes in the cholesterol biosynthesis/homeostasis pathway that were completely reverted with LDL pretreatment. In silico analysis of patient data supported a role for cholesterol in response to bortezomib-based therapies. Targeted proteome profiling revealed changes in expression of the adipokine resistin in the bone marrow of cholesterol-treated or myeloma-bearing mice, with elevated resistin expression observed in MGUS patients. Bone marrow adipocytes were found to be a major source of resistin, which was further increased in response to LDL. In summary, we demonstrate that high cholesterol promotes both myeloma development and bortezomib resistance, identifying resistin as a potential mediator so revealing new mechanisms underlying myeloma pathogenesis. ### Competing Interest Statement The authors have declared no competing interest.

Antibody engineering can tailor the design and activities of therapeutic antibodies for better efficiency or other advantageous clinical properties. Here we report the development of ISB 1442, a fully human bispecific antibody designed to re-establish synthetic immunity in CD38+ hematological malignancies. ISB 1442 consists of two anti-CD38 arms targeting two distinct epitopes that preferentially drive binding to tumor cells and enable avidity-induced blocking of proximal CD47 receptors on the same cell while preventing on-target off-tumor binding on healthy cells. The Fc portion of ISB 1442 is engineered to enhance complement dependent cytotoxicity, antibody dependent cell cytotoxicity and antibody dependent cell phagocytosis. ISB 1442 thus represents a CD47-BsAb combining biparatopic targeting of a tumor associated antigen with engineered enhancement of antibody effector function to overcome potential resistance mechanisms that hamper treatment of myeloma with monospecific anti-CD38 antibodies. ISB 1442 is currently in a Phase I clinical trial in relapsed refractory multiple myeloma.

Abstract Immune therapies targeting single tumor associated antigens (TAA) demonstrated efficacy against multiple myeloma (MM). However, durable responses are still limited1 potentially due to expansion of clones with low target expression2 We have previously demonstrated that simultaneous targeting of BCMA and CD38 on MM tumor cells with heterogenous expression of these antigens using dual targeting ISB 2001, enables superior killing relative to mono-targeting T cell engagers (TCE) 3. Here we present a further characterization of a dual targeting TCE utilizing bone marrow aspirates from multiple myeloma patients. In relapsed/refractory (r/r) patients, which received CD38 targeted therapy, daratumumab cytotoxicity was substantially reduced due to low CD38 expression. Teclistamab is considered the next line of treatment in such patients. However, ISB 2001 consistently demonstrated increased cytotoxicity compared to teclistamab in both newly diagnosed patient and r/r patient samples. Remarkably, ISB 2001 also induced stronger cytotoxic response in one patient relapsing after BCMA targeted therapy, suggesting that the dual targeting ISB 2001 TCE can overcome the escape mechanisms. ISB 2001 was also compared to the combination of teclistamab and daratumumab in cytotoxic assay using healthy donors. ISB 2001 shows superiority to both teclistamab and daratumumab as single agents, as well as when they were used in combination. Additionally, ISB 2001 was compared against a combination of daratumumab and teclistamab in a humanized mouse model of multiple myeloma with a low expression of both CD38 and BCMA, mimicking potential tumor escaping clones. ISB 2001 induced complete eradication of the tumors in almost all animals, whereas the combination only showed partial tumor protection, underscoring that dual targeting by a TCE is superior to two therapeutic agents individually targeting the same antigens. Based on the promising preclinical in vitro, ex vivo and in vivo data, we have advanced ISB 2001 into clinical studies. For calculating the first-in-human (FIH) dose, we have developed a quantitative systems pharmacology (QSP) model. The calculated FIH dose was 50-100 fold higher than that generated by a traditional minimum anticipated biological effect level calculation. Therefore, using this approach will substantially reduce patient exposure to sub-efficacious doses of ISB 2001. This plan was accepted by the HREC in Australia and the FDA in the US to initiate a Phase 1 FIH study of ISB 2001 for the treatment of relapsed/refractory multiple myeloma (NCT05862012)4. 1. Munshi, N. C. et al. 384, 705-716 (2021). 2. Nijhof, I. S. et al. Blood 128, 12 (2016). 3. (https://doi.org/10.1182/blood-2022-159353) 4. Abstract# 3396, ASH 2023, Hanlon Sia at al. Citation Format: Laura Carretero-Iglesia, Maria Pihlgren, Jeremy Berret, Adam Drake, Daniela Pais, Cyrille Dreyfus, Vinu Menon, Thomas Matthes, Claire Edwards, James Edwards, Catherine Pellat-Deceunynck, Philippe Moreau, Cyrille Touzeau, Tomomi Matsuura, Piet van der Graaf, Lida Pacaud, Cyril Konto, Eugene Zhukovsky, Mario Perro. ISB 2001, a BCMA and CD38 dual targeting T cell engager, demonstrates superior cytotoxicity relative to teclistamab in the samples of patient relapsing from CD38 and BCMA targeted immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1238.

Despite recent advances in immunotherapies targeting single tumor-associated antigens, patients with multiple myeloma eventually relapse. ISB 2001 is a CD3+ T cell engager (TCE) co-targeting BCMA and CD38 designed to improve cytotoxicity against multiple myeloma. Targeting of two tumor-associated antigens by a single TCE resulted in superior cytotoxic potency across a variable range of BCMA and CD38 tumor expression profiles mimicking natural tumor heterogeneity, improved resistance to competing soluble factors and exhibited superior cytotoxic potency on patient-derived samples and in mouse models. Despite the broad expression of CD38 across human tissues, ISB 2001 demonstrated a reduced T cell activation profile in the absence of tumor cells when compared to TCEs targeting CD38 only. To determine an optimal first-in-human dose for the ongoing clinical trial (NCT05862012), we developed an innovative quantitative systems pharmacology model leveraging preclinical data, using a minimum pharmacologically active dose approach, therefore reducing patient exposure to subefficacious doses of therapies. Perro and colleagues develop a CD3+ T cell engager co-targeting BCMA and CD38 to improve immunotherapy for multiple myeloma, demonstrate cytotoxicity in patient-derived samples and murine models and develop a quantitative systems pharmacology model.

Circulating tumour cells (CTCs) are cancer cells shed from a primary tumour which intravasate into the blood stream and have the potential to extravasate into distant tissues, seeding metastatic lesions. As such, they can offer important insight into cancer progression with their presence generally associated with a poor prognosis. The detection and enumeration of CTCs is, therefore, critical to guiding clinical decisions during treatment and providing information on disease state. CTC isolation has been investigated using a plethora of methodologies, of which immunomagnetic capture and microfluidic size-based filtration are the most impactful to date. However, the isolation and detection of CTCs from whole blood comes with many technical barriers, such as those presented by the phenotypic heterogeneity of cell surface markers, with morphological similarity to healthy blood cells, and their low relative abundance (∼1 CTC/1 billion blood cells). At present, the majority of reported methods dissociate CTC isolation from detection, a workflow which undoubtedly contributes to loss from an already sparse population. This review focuses on developments wherein isolation and detection have been integrated into a single-step, microfluidic configuration, reducing CTC loss, increasing throughput, and enabling an on-chip CTC analysis with minimal operator intervention. Particular attention is given to immune-affinity, microfluidic CTC isolation, coupled to optical, physical, and electrochemical CTC detection (quantitative or otherwise).

Immune therapies targeting a single tumor associated antigen (TAA) have demonstrated their efficacy against multiple myeloma (MM) in recent years. However, durable responses are still limited, which may be due to downregulation of the targeted TAA or expansion of clones with low target expression . To improve binding to tumor cells and enhance tumor cell killing, we propose to simultaneously target BCMA and CD38 using ISB 2001, a potential first-in-class TREAT™ trispecific CD3 T-cell engager based on Ichnos’ proprietary BEAT platform. To understand whether dual targeting could improve binding to tumors and enhance killing, we compared ISB 2001 to control molecules lacking one of the two TAA binders. Potency of ISB 2001 was greater than control molecules lacking either BCMA or CD38 used alone or in combination in a re-directed lysis assay on multiple myeloma cells These results were consistent with the binding to tumor cell lines, in which ISB 2001 showed higher maximal binding than control molecules lacking either CD38 or BCMA binding domains. We also compared the tumor killing potency of ISB 2001 to teclistamab, a BCMAxCD3 bispecific antibody recently approved to treat relapsed/refractory (RR) MM patients who have received prior lines of treatment. To mimic the heterogeneity of tumor cells, the killing of MM cell lines expressing varying levels of BCMA and CD38 was evaluated. ISB 2001 exhibited potent killing of the cell lines with EC50 ranging from 0.2 to 1.5 pM, which was statistically superior to that of teclistamab. These results were consistent with elevated binding of ISB 2001 to MM cells compared to teclistamab. In the presence of soluble BCMA or APRIL, ISB 2001 was 100-fold more potent than teclistamab. When evaluated in a therapeutic PBMC-humanized mouse model subcutaneously engrafted with BCMAlow/CD38low KMS-12-BM cells, ISB 2001 resulted in complete tumor regression and showed statistically higher potency than teclistamab at 0.1 mg/kg dose. In addition, ISB 2001 showed more potent anti-tumor activity than teclistamab in bone marrow aspirates of MM patients, indicating that ISB 2001 to leverage its cytotoxic properties with available immune cells. Taken together, we show that there is a clear advantage of avid binding achieved by the dual targeting using a TREAT-based ISB 2001, which results in potent and efficacious killing of tumor cells in vitro and in vivo. These data support clinical development of ISB 2001 as a promising treatment of RRMM through co-targeting of BCMA and CD38. Significant benefit is anticipated for RRMM patients who may experience tumor escape through target downregulation mechanisms. The evaluation of ISB2001 in a phase 1 clinical trial is planned for the first half of 2023. Citation Format: Maria Pihlgren, Laura Carretero, Jeremy Berret, Olivia Hall, Carole Estoppey, Adam Drake, Daniela Pais, Julie Macoin, Myriam Chimen, Perrine Suere, Emily Nallet, Blandine Pouleau, Elodie Stainnack, Jeremy Loyau, Thierry Monney, Aurore Delachat, Cyrille Dreyfus, Stan Blein, Zeynep Kaya, Thomas Matthes, Rebecca Croasdale-wood, James Edwards, Claire Edwards, Lamine Mbow, Michael Dyson, Cyril Konto, Ankita Srivastava, Eugene Zhukovshy, Mario Perro. Overcoming mechanisms of escape from treatments for multiple myeloma with ISB 2001, a potential first-in-class trispecific BCMA and CD38 targeted T cell engager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2970.

Novel biomarkers for tumour burden and bone disease are required to guide clinical management of plasma cell dyscrasias. Recently, bone turnover markers (BTMs) and Diffusion-Weighted Magnetic Resonance Imaging (DW-MRI) have been explored, although their role in the prospective assessment of multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) is unclear. Here, we conducted a pilot observational cohort feasibility study combining serum BTMs and DW-MRI in addition to standard clinical assessment. Fifty-five patients were recruited (14 MGUS, 15 smouldering MM, 14 new MM and 12 relapsed MM) and had DW-MRI and serum biomarkers (P1NP, CTX-1, ALP, DKK1, sclerostin, RANKL:OPG and BCMA) measured at baseline and 6-month follow-up. Serum sclerostin positively correlated with bone mineral density (r = 0.40−0.54). At baseline, serum BCMA correlated with serum paraprotein (r = 0.42) and serum DKK1 correlated with serum free light chains (r = 0.67); the longitudinal change in both biomarkers differed between International Myeloma Working Group (IMWG)-defined responders and non-responders. Myeloma Response Assessment and Diagnosis System (MY-RADS) scoring of serial DW-MRI correlated with conventional IMWG response criteria for measuring longitudinal changes in tumour burden. Overall, our pilot study suggests candidate radiological and serum biomarkers of tumour burden and bone loss in MM/MGUS, which warrant further exploration in larger cohorts to validate the findings and to better understand their clinical utility.

Lipids play a major role in inflammatory diseases by altering inflammatory cell functions, either through their function as energy substrates or as lipid mediators such as oxylipins. Autophagy, a lysosomal degradation pathway that limits inflammation, is known to impact on lipid availability, however, whether this controls inflammation remains unexplored. We found that upon intestinal inflammation visceral adipocytes upregulate autophagy and that adipocyte‐specific loss of the autophagy gene Atg7 exacerbates inflammation. While autophagy decreased lipolytic release of free fatty acids, loss of the major lipolytic enzyme Pnpla2/Atgl in adipocytes did not alter intestinal inflammation, ruling out free fatty acids as anti‐inflammatory energy substrates. Instead, Atg7 ‐deficient adipose tissues exhibited an oxylipin imbalance, driven through an NRF2‐mediated upregulation of Ephx1 . This shift reduced secretion of IL‐10 from adipose tissues, which was dependent on the cytochrome P450‐EPHX pathway, and lowered circulating levels of IL‐10 to exacerbate intestinal inflammation. These results suggest an underappreciated fat‐gut crosstalk through an autophagy‐dependent regulation of anti‐inflammatory oxylipins via the cytochrome P450‐EPHX pathway, indicating a protective effect of adipose tissues for distant inflammation.

Regions of hypoxia occur in most tumors and predict for poor patient prognosis. Hypoxia-activated prodrugs provide an ideal strategy to target the aggressive, hypoxic fraction of a tumor while protecting the normal tissue from toxicity. A key challenge associated with the development of novel hypoxia-activated prodrugs, however, is the ability to visualize the delivery of the prodrug to hypoxic regions and determine where it has been activated. Here we report a modified version of the commonly used nitroimidazole bioreductive group that incorporates the fluoroethyl epitope of the antibody-based hypoxia imaging agent, EF5. Attachment of this group to the red fluorescent dye, DCM, enabled us to correlate release of the DCM dye with imaging of the reduced bioreductive group using the EF5 antibody. This study confirmed that the antibody was imaging reduction and fragmentation of the pro-fluorophore. We next employed the modified bioreductive group to synthesize a new prodrug of the KDAC inhibitor Panobinostat, EF5-Pano. Release of EF5-Pano in hypoxic multiple myeloma cells was imaged using the EF5 antibody, and the presence of an imaging signal correlated with apoptosis and a reduction in cell viability. Therefore, EF5-Pano is an imageable hypoxia-activated prodrug with proven cytotoxic effect in multiple myeloma, which could be utilized in future in vivo experiments.

Whilst treatment of multiple myeloma (MM) with daratumumab significantly extend patient lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/rMM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T-cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT®) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38 including those with a lower sensitivity to daratumumab. In a killing assay, wherein multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity towards MM cells compared to daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in two therapeutic mouse models, unlike daratumumab. Lastly, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/rMM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently developed in a phase 1 clinical study.

Multiple myeloma is a B cell neoplasm caused by the monoclonal expansion and infiltration of malignant plasma cells in the bone marrow. Myeloma growth can significantly disrupt normal bone function by cell-to-cell interactions and by the secretion of factors that promote osteoclastic bone resorption and inhibit osteoblastic bone formation. This imbalance can lead to the development of osteolytic lesions, hypercalcemia, and susceptibility to bone fractures. Ultimately a vicious cycle is created that increases bone damage, releasing more factors that in turn enhance myeloma cell growth. In addition, other cells within the bone marrow microenvironment, including osteocytes, adipocytes, and immune cells, can influence myeloma cell colonization, survival, and subsequent growth or dormancy. Myeloma in vivo models, patient studies, and clinical trials support these findings. In this chapter we will discuss the biological relationship between cells in the bone marrow microenvironment and myeloma cells, describing key mechanisms that disrupt normal bone function and current bone modulating therapies.