Belantamab mafodotin induces immunogenic cell death within 24 h post‐administration in newly diagnosed multiple myeloma patients

Despite the outstanding progress in the clinical management of Multiple Myeloma (MM) over the last decades, the disease remains incurable with unavoidable patients' relapses. In this context, there is a constant need for the development of novel therapeutic regimens that will ensure a prolonged clinical and molecular remission of the disease overcoming current treatment refractoriness. In August 2020, the FDA issued regulatory approval for an innovative drug designated for relapsed/refractory MM (RRMM) patients, who have previously received at least four lines of therapy, including a proteasome inhibitor (PI), an immunomodulatory drug (IMiD) as well as a monoclonal antibody (mAb) against CD38.1 The new agent Belantamab Mafodotin (Βelamaf; Blenrep®), is a first-in-class antibody-drug conjugate (ADC) composed of an afucosylated IgG1 mAb against B-cell maturation antigen (BCMA), conjugated to the microtubule-disrupting agent monomethyl auristatin F (MMAF; mafodotin) via an uncleavable maleimidocaproyl (mc) cysteine linker region, which renders the ADC stable in circulation.2 Belamaf contributes to the depletion of aberrant plasma cells through a multimodal mechanism of action that includes direct cell killing via the rapid release of the cytotoxic MMAF inside the BCMA-expressing myeloma cells, antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cell-mediated cytotoxicity (ADCC).2 Recent in vitro and in vivo evidence in mice support that Belamaf promotes anti-myeloma activity through the induction of immunogenic cell death (ICD),3 a type of regulated cell death (RCD), which may trigger immune stimulation by endogenous components released from dying or dead cells.4 ICD is characterized by the extracellular release of danger-associated molecular patterns (DAMPs, else termed alarmins), endogenous immunogenic compounds which, in the presence of tumor antigens, act as adjuvants in the microenvironment of dying cells, and, after their binding to innate immune receptors on antigen-presenting cells (APC), they likely promote tumor antigen-reactive T cell immune responses.5 Well-known DAMPs with immunostimulatory properties are calreticulin (CRT), the high mobility group box 1 protein (HMGB1), and prothymosin α (proTα) or its immunopotent fragment proTα(100–109). CRT is normally localized in the endoplasmic reticulum (ER) and is responsible for calcium ion (Ca+2) homeostasis, as it binds 50% of total cell calcium within the ER. In the early phases of ICD, CRT translocates to the cell membrane, where it acts as an "eat me" signal for phagocytes.4, 5 HMGB1 and proTα are nuclear proteins, which, in normal conditions, interact with various transcription factors and histones, having a key role in chromatin remodeling and transcription regulation. However, at later stages of ICD, extracellularly released HMGB1 may be captured by APCs, triggering the production of both proinflammatory and inflammatory cytokines.4-6 Similarly, during ICD, the truncated by activated caspases carboxy-terminal decapeptide proTα(100–109) is released, binds Toll-like receptor 4 on APCs and consequently stimulates T cell activation.7, 8 Herein, we evaluated for the first time the actual effect of ICD in newly diagnosed MM (NDMM) patients treated with Belamaf, by measuring the levels of the three DAMPs prior and after Belamaf administration. In specific, 15 transplant-ineligible NDMM patients enrolled in the BelaRD clinical trial (NCT04808037), received two cycles of Belamaf with an interval of 60 days, as part of the induction therapy, which also included dexamethasone and lenalidomide. All DAMPs were evaluated in the peripheral blood (PB) of treated patients at two consecutive time-points for both cycles, prior [day 0 (baseline levels) and day 60] and 24 h after Belamaf administration (days 1 and 61). The levels of HMGB1 were estimated in patients' serum with a commercially available highly-sensitive ELISA (Cloud-Clone Corp. TX), whereas the levels of proTα(100–109) were evaluated in patients' plasma using an in-house competitive ELISA.4, 8 The expression of CRT was evaluated on the surface of circulating clonal plasma cells (CTCs) with flow cytometry, be adding an APC-conjugated rabbit anti-human CRT antibody (EPR3924, Abcam, Cambridge, UK) and its relevant isotypic control, to a multiparametric panel containing fluorochrome-conjugated antibodies against human CD38 (FITC; Cytognos, Salamanca, Spain), CD45 (PerCPCy5.5; BD, NJ), CD56 (PE; Cytognos), CD138 (BV421; BD) and CD19 (PeCy7; Beckman Coulter, CA), which allows for the effective discrimination of CTCs, B, T, NK cells, monocytes and neutrophils among total PB nucleated cells as previously described.9 Belamaf was efficient as early as at 24 h post-administration, as evidenced by the rigorous decrease in the numbers of CTCs. All patients had detectable CTCs at day 0 (median value 0.015% of total PB nucleated cells), but CTCs were detected in 12/15 patients (80%) at day 1, with a significant shrinkage in their burden (median value 0.0008%) (Figure S1). Of note, CTCs became undetectable for 14/15 patients (93%) after the second Belamaf cycle, with only one patient showing remaining CTCs which were reduced by three logarithmic units as compared with baseline levels. Patients' PB B cells followed the same kinetics, as their numbers were significantly reduced post-Belamaf administration and remained at very low levels during the entire study period. On the contrary, the percentages of T cells, NK cells, and monocytes were only transiently decreased after Belamaf administration, possibly due to a temporal and BCMA-irrelevant side effect, and recovered to their baseline levels before the second cycle (Figure S2). We further analyzed whether the significant reduction of CTCs within 24 h following Belamaf administration was due to ICD induced in vivo by the ADC. Indeed, mean fluorescent intensity (MFI) of CRT on CTCs was almost equal with the expression of the isotypic control on day 0, but the ratio (MFI of CRT to the MFI of isotype) showed a robust increase on day 1, highlighting that the remaining CTCs expressed significantly higher levels of CRT after Belamaf administration (Figure 1A). Similarly, the consecutive analysis of patients' sera revealed a 3-fold increase of HMGB1 concentration at 24 h post-Belamaf administration (mean value 55 ng/mL vs. 165 ng/mL, on days 0 and 1, respectively; p < 0.0001, Figure 1B). The levels of proTα(100–109) had also a clear trend of increase after treatment (mean values 2.6 ng/mL prior vs. 3.2 ng/mL post-Belamaf; p = 0.08; Figure 1C), further supporting the anti-myeloma efficacy of Belamaf through the induction of ICD. The latter differences were less prominent compared to the robust increase of HMGB1, possibly implying, that at the time of the (second) sampling (24 h post-Belamaf) only DAMPs released at later stages of ICD (like HMGB1) could be clearly detected. In clinical terms, patients displaying a favorable three-month response to induction therapy (very good partial response or better) had more profound evidence of ICD than those with partial response, and could be sufficiently discriminated in a PCA model estimating the 24 h changes of HMGB1 and proΤα(100–109) due to Belamaf administration (Figure 1D). Altogether, Belamaf is an effective drug against BCMA-expressing cells with promising potential for use at various lines of MM treatment. Our study provides clear evidence that the drug acts rapidly (within 24 h) and promotes in vivo the ICD of myeloma cells. Moreover, our results support an association between the intensity of ICD induction early during therapy with the subsequent response-to-treatment, which could prove particularly useful for an efficient prompt stratification and subsequent monitoring of patients receiving modern treatments of Belamaf combined with novel agents.10 Evangelos Terpos, Efstathios Kastritis, Ourania Tsitsilonis, and Meletios-Athanasios Dimopoulos designed the research; Ioannis V. Kostopoulos, Antonis Kakalis, Anastasios Birmpilis, and Ourania Tsitsilonis performed laboratory analyses; Evangelos Terpos, Maria Gavriatopoulou, Chrysanthi Panteli, Pantelis Rousakis, Nikolaos Angelis, Efstathios Kastritis, Nikolaos Orologas-Stavrou and Meletios A. Dimopoulos collected samples and patients' data; Ioannis V. Kostopoulos, Antonis Kakalis, Anastasios Birmpilis and Evangelos Terpos analyzed and interpreted data; Ioannis V. Kostopoulos and Antonis Kakalis performed statistical analysis; Ioannis V. Kostopoulos, Antonis Kakalis, and Ourania Tsitsilonis wrote the manuscript. All authors reviewed and agreed to the final version of the manuscript. This work was partly funded by the GSRT Operational program "Competitiveness, Entrepreneurship, & Innovation (EPAnEK)" 2014–2020. Evangelos Terpos, Efstathios Kastritis, Maria Gavriatopoulou and Meletios A. Dimopoulos have received honoraria and research support by GSK; all other authors declare that there is no conflict of interest relevant to this research. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1. Figure S2. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.