Integrative Analysis of Proteomics and Transcriptomics Reveals Endothelin Receptor B as Novel Single Target and Identifies New Combinatorial Targets for Multiple Myeloma.

Despite the recent introduction of next-generation immunotherapeutic agents, multiple myeloma (MM) remains incurable. New strategies targeting MM-specific antigens may result in a more effective therapy by preventing antigen escape, clonal evolution, and tumor resistance. In this work, we adapted an algorithm that integrates proteomic and transcriptomic results of myeloma cells to identify new antigens and possible antigen combinations. We performed cell surface proteomics on 6 myeloma cell lines based and combined these results with gene expression studies. Our algorithm identified 209 overexpressed surface proteins from which 23 proteins could be selected for combinatorial pairing. Flow cytometry analysis of 20 primary samples confirmed the expression of FCRL5, BCMA, and ICAM2 in all samples and IL6R, endothelin receptor B (ETB), and SLCO5A1 in >60% of myeloma cases. Analyzing possible combinations, we found 6 combinatorial pairs that can target myeloma cells and avoid toxicity on other organs. In addition, our studies identified ETB as a tumor-associated antigen that is overexpressed on myeloma cells. This antigen can be targeted with a new monoclonal antibody RB49 that recognizes an epitope located in a region that becomes highly accessible after activation of ETB by its ligand. In conclusion, our algorithm identified several candidate antigens that can be used for either single-antigen targeting approaches or for combinatorial targeting in new immunotherapeutic approaches in MM.

Radiotheranostic Agents in Hematological Malignancies.

Radioimmunotherapy (RIT) is a cancer treatment that combines radiation therapy with tumor-directed monoclonal antibodies (Abs). Although RIT had been introduced for the treatment of CD20 positive non-Hodgkin lymphoma decades ago, it never found a broad clinical application. In recent years, researchers have developed theranostic agents based on Ab fragments or small Ab mimetics such as peptides, affibodies or single-chain Abs with improved tumor-targeting capacities. Theranostics combine diagnostic and therapeutic capabilities into a single pharmaceutical agent; this dual application can be easily achieved after conjugation to radionuclides. The past decade has seen a trend to increased specificity, fastened pharmacokinetics, and personalized medicine. In this review, we discuss the different strategies introduced for the noninvasive detection and treatment of hematological malignancies by radiopharmaceuticals. We also discuss the future applications of these radiotheranostic agents.

A non-internalised CD38-binding radiolabelled single-domain antibody fragment to monitor and treat multiple myeloma.

BACKGROUND: Antibody-based therapies targeting CD38 are currently used as single agents as well as in combination regimens for multiple myeloma, a malignant plasma cell disorder. In this study, we aimed to develop anti-CD38 single-domain antibodies (sdAbs) that can be used to trace CD38+ tumour cells and subsequently used for targeted radionuclide therapy. SdAbs are derived from Camelidae heavy-chain antibodies and have emerged as promising theranostic agents due to their favourable pharmacological properties. METHODS: Four different anti-CD38 sdAbs were produced, and their binding affinities and potential competition with the monoclonal antibody daratumumab were tested using biolayer interferometry. Their binding kinetics and potential cell internalisation were further studied after radiolabelling with the diagnostic radioisotope Indium-111. The resulting radiotracers were evaluated in vivo for their tumour-targeting potential and biodistribution through single-photon emission computed tomography (SPECT/CT) imaging and serial dissections. Finally, therapeutic efficacy of a lead anti-CD38 sdAb, radiolabelled with the therapeutic radioisotope Lutetium-177, was evaluated in a CD38+ MM xenograft model. RESULTS : We retained anti-CD38 sdAb #2F8 as lead based on its excellent affinity and superior stability, the absence of competition with daratumumab and the lack of receptor-mediated internalisation. When intravenously administered to tumour-xenografted mice, radiolabelled sdAb #2F8 revealed specific and sustained tumour retention with low accumulation in other tissues, except kidneys, resulting in high tumour-to-normal tissue ratios. In a therapeutic setting, myeloma-bearing mice received three consecutive intravenous administrations of a high (18.5 MBq) or a low radioactive dose (9.3 MBq) of 177Lu-DTPA-2F8 or an equal volume of vehicle solution. A dose-dependent tumour regression was observed, which translated into a prolonged median survival from 43 days for vehicle-treated mice, to 62 days (p = 0.027) in mice receiving the low and 65 days in mice receiving the high (p = 0.0007) radioactive dose regimen, respectively. CONCLUSIONS: These results highlight the theranostic potential of radiolabelled anti-CD38 sdAbs for the monitoring and treatment of multiple myeloma.

Balancing the CD38 Expression on Effector and Target Cells in Daratumumab-Mediated NK Cell ADCC against Multiple Myeloma.

Multiple myeloma (MM) is an incurable cancer characterized by the proliferation and accumulation of monoclonal plasma cells in the bone marrow. The monoclonal anti-CD38 daratumumab has taken a central place in the different treatment regimens for newly diagnosed and relapsed, refractory myeloma. In this study, we correlated the NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and potential fratricide induced by daratumumab with CD38-expression levels on both effector and target cells. We show that CD38 expression can be modulated by adding all-trans retinoic acid (ATRA) or interferon-α to MM cells to further fine-tune these effects. In addition, we observed that ADCC becomes inefficient when fratricide occurs and both ADCC and fratricide depend on the balance between CD38 expression on effector and target cells. However, the addition of adjuvants (retinoic acid or interferon-α) to myeloma cells or the inhibition of fratricide using a CD38-blocking nanobody on NK-cells can reverse this balance towards ADCC and thus promote lysis of target cells by ADCC. ATRA and interferon-α increased the CD38 expression at the surface of MM cells about three-fold and two-fold, respectively. This increase was of interest for MM cells with low CD38 expression, that became susceptible to daratumumab-mediated ADCC after preincubation. A CD38-blocking nanobody prevented the binding of daratumumab to these NK-cells and blunted the fratricidal effect on effector NK cells. In conclusion, our study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated ADCC.

Bispecific, T-Cell-Recruiting Antibodies in B-Cell Malignancies.

Bispecific antibodies (BsAbs) are designed to recognize and bind to two different antigens or epitopes. In the last few decades, BsAbs have been developed within the context of cancer therapies and in particular for the treatment of hematologic B-cell malignancies. To date, more than one hundred different BsAb formats exist, including bispecific T-cell engagers (BiTEs), and new constructs are constantly emerging. Advances in protein engineering have enabled the creation of BsAbs with specific mechanisms of action and clinical applications. Moreover, a better understanding of resistance and evasion mechanisms, as well as advances in the protein engineering and in immunology, will help generating a greater variety of BsAbs to treat various cancer types. This review focuses on T-cell-engaging BsAbs and more precisely on the various BsAb formats currently being studied in the context of B-cell malignancies, on ongoing clinical trials and on the clinical concerns to be taken into account in the development of new BsAbs.

The anti-mitotic agents PTC-028 and PTC596 display potent activity in pre-clinical models of multiple myeloma but challenge the role of BMI-1 as an essential tumour gene.

Future progress in the treatment of multiple myeloma (MM) requires both the characterisation of key drivers of the disease and novel, innovative approaches to tackle these vulnerabilities. The present study focussed on the pre-clinical evaluation of a novel drug class, BMI-1 modulators, in MM. We demonstrate potent activity of PTC-028 and PTC596 in a comprehensive set of in vitro and in vivo models, including models of drug resistance and stromal support. Treatment of MM cells with PTC-028 and PTC596 downregulated BMI-1 protein levels, which was found to correlate with drug activity. Surprisingly, BMI-1 was dispensable for the activity of BMI-1 modulators and MM cell growth. Our data rather point to mitotic arrest accompanied by myeloid cell leukaemia-1 (MCL-1) loss as key anti-MM mechanisms and reveal impaired MYC and AKT signalling activity due to BMI-1 modulator treatment. Moreover, we observed a complete eradication of MM after PTC596 treatment in the 5TGM.1 in vivo model and define epigenetic compounds and B cell leukaemia/lymphoma 2 homology domain 3 (BH3) mimetics as promising combination partners. These results bring into question the postulated role of BMI-1 as an essential MM gene and confirm BMI-1 modulators as potent anti-mitotic agents with encouraging pre-clinical activity that supports their rapid translation into clinical trials.

Loss of Stromal Galectin-1 Enhances Multiple Myeloma Development: Emphasis on a Role in Osteoclasts.

Multiple myeloma osteolytic disease is caused by an uncoupled bone-remodelling process with an increased osteoclast activity. Disease development relies on interactions between myeloma cells and bone marrow stromal cells. Recent findings suggest a role for glycan-binding proteins in myeloma microenvironment. Here, we investigated lectins involved in osteoclastogenesis and their role in myeloma bone disease. Microarray data analysis showed a lower expression of galectin-1 (gal-1) in mature osteoclasts compared to monocytic progenitor cells, confirmed at the RNA and protein levels in osteoclast cultures. Confocal microscopy showed that gal-1 localised predominantly in the sealing zone of mature osteoclasts. Although equal differentiated-osteoclast numbers, gal-1-/- osteoclasts showed a higher resorption activity compared to wild-type controls. Micro-computed tomography showed an aberrant bone phenotype with decreased bone densities in gal-1-/- mice. In vivo, tumour progression was faster in gal-1-/- mice and associated with a marked bone loss. Additionally, myeloma cells were found to decrease gal-1 expression in osteoclasts. Our results demonstrate that galectin-1 regulates osteoclast activity with an increased resorption by gal-1-/- osteoclasts and decreased bone densities in gal-1-/- mice. We observed an enhanced tumour development in gal-1-/- mice compared to wild-type mice, suggesting that galectin-1 has a functional role in stromal cells in myeloma microenvironment.

Maternal embryonic leucine zipper kinase inhibitor OTSSP167 has preclinical activity in multiple myeloma bone disease.

Multiple myeloma bone disease is characterized by an uncoupling of bone remodeling in the multiple myeloma microenvironment, resulting in the development of lytic bone lesions. Most myeloma patients suffer from these bone lesions, which not only cause morbidity but also negatively impact survival. The development of novel therapies, ideally with a combined anti-resorptive and bone-anabolic effect, is of great interest because lesions persist with the current standard of care, even in patients in complete remission. We have previously shown that MELK plays a central role in proliferation-associated high-risk multiple myeloma and its inhibition with OTSSP167 resulted in decreased tumor load. MELK inhibition in bone cells has not yet been explored, although some reports suggest that factors downstream of MELK stimulate osteoclast activity and inhibit osteoblast activity, which makes MELK inhibition a promising therapeutic approach. Therefore, we assessed the effect of OTSSP167 on bone cell activity and the development of myeloma-induced bone disease. OTSSP167 inhibited osteoclast activity in vitro by decreasing progenitor viability as well as via a direct anti-resorptive effect on mature osteoclasts. In addition, OTSSP167 stimulated matrix deposition and mineralization by osteoblasts in vitro This combined anti-resorptive and osteoblast-stimulating effect of OTSSP167 resulted in the complete prevention of lytic lesions and bone loss in myeloma-bearing mice. Immunohistomorphometric analyses corroborated our in vitro findings. In conclusion, we show that OTSSP167 has a direct effect on myeloma-induced bone disease in addition to its anti-multiple myeloma effect, which warrants further clinical development of MELK inhibition in multiple myeloma.

Molecular mechanisms, current management and next generation therapy in myeloma bone disease.

Multiple myeloma (MM) bone disease is a major cause of morbidity and mortality in MM patients and persists even in patients in remission. This bone disease is caused by an uncoupling of bone remodeling, with increased osteoclast and decreased osteoblast activity and formation, culminating in lytic bone destruction. Bisphosphonates are the current standard of care but new therapies are needed. As the molecular mechanisms controlling MM bone disease are increasingly well understood, new therapeutic targets are extensively explored in the preclinical setting and initial clinical trials with novel compounds now show promising results. In this review, we will provide a comprehensive overview of the biology of MM bone disease, summarize its current clinical management and discuss preclinical and clinical data on next generation therapies.