Killing Of Multiple Myeloma Cells Research Articles

Comparative performance of scFv-based anti-BCMA CAR formats for improved T cell therapy in multiple myeloma

In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.

Abstract 1322: A cryopreserved allogeneic anti-BCMA CAR-NK cellular therapy exhibits both innate and CAR-mediated MM cell killing in vitro and in vivo

Abstract Introduction Multiple myeloma (MM) is the second most prevalent hematopoietic malignancy with an overall 5-year survival rate of 58%. B cell maturation antigen (BCMA) is selectively expressed on normal and malignant plasma cells, making it an attractive MM target. Anti-BCMA chimeric antigen receptor (CAR) T cell therapy and T cell engagers have shown promise as treatment options for certain MM patients; however, potential toxicities associated with these anti-BCMA therapies, such as cytokine release syndrome (CRS), have limited broader utilization. Much like T cells, natural killer (NK) cells are cytolytic and have demonstrated an innate capacity to reduce tumor burden while exhibiting a more favorable safety profile in clinical testing. To that end, we developed a cryopreserved allogeneic cell therapy comprised of genetically modified human umbilical cord blood-derived (CB) NK cells transduced with a gammaretroviral vector, which incorporates genes for an anti-BCMA CAR and soluble human interleukin-15 (sIL-15). Referred to hereafter as anti-BCMA CAR-NK, it exhibits both innate NK- and CAR-mediated killing in vitro and robust in vivo activity against established MM tumors. Methods CBNK cells were isolated from donor cord blood units, propagated using feeder cells, transduced with a gammaretroviral vector to express an anti-BCMA CAR and soluble human IL-15, and propagated further before harvest and cryopreservation. Donor-equivalent untransduced (UTD) NK cells were generated via the same process but without the transduction step. Cryopreserved anti-BCMA CAR-NK and UTD NK cells were used for in vitro studies, including short-term killing, as well as for evaluation of activity against established MM tumors in vivo using the MM.1S-Luc4 model. Results The anti-BCMA CAR was successfully expressed across all batches of anti-BCMA CAR-NK generated. Results from an in vitro cytotoxicity assay indicated that anti-BCMA CAR-NK kills BCMA expressing MM.1S-Luc4 tumor cells and secretes IFNγ, TNFα, and granzyme B at greater amounts compared to donor-equivalent UTD NK. Anti-BCMA CAR-NK and UTD NK cells also demonstrated equivalent cytotoxicity towards BCMA non-expressing cell lines, JJN3-Luc BCMA KO and NCI-H520, in an effector:target cancer cell dependent manner, highlighting the potential for treatment with anti-BCMA CAR-NK post-prior BCMA therapy. In in vivo studies, anti-BCMA CAR-NK exhibited robust anti-tumor activity in an MM.1S-Luc4 NSG mouse xenograft model, with no signs of anti-BCMA CAR-NK related body weight loss. Conclusion A cryopreserved allogeneic anti-BCMA CAR-NK cellular therapy exhibits both innate and CAR-mediated killing in vitro and robust in vivo activity in MM tumor models. Preclinical data supports future clinical evaluation in relapsed/refractory MM patients who have received prior BCMA therapy and IND enabling studies are ongoing. Citation Format: LeeAnn Talarico, Christina Wong, Chunyan Pang, Taylor Hickman, Chenqi Hu, Amy Shaw, Emily Wisniewski, Shao-Chiang (Michael) Lai, Pranjal Sharma, Shaun Moore, Luan Nguyen, Kayla Rhuda, Saurin Patel, Paul Lin, Rafet Basar, Shawn Cogan, Kat Sofjan, Arun Ramamurthy, Aaron Handler, Kathryn Fraser, Yana Wang, Katayoun Rezvani, Michael D. Curley. A cryopreserved allogeneic anti-BCMA CAR-NK cellular therapy exhibits both innate and CAR-mediated MM cell killing in vitro and in vivo [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 1322.

CD46 and CD59 inhibitors enhance complement-dependent cytotoxicity of anti-CD38 monoclonal antibodies daratumumab and isatuximab in multiple myeloma and other B-cell malignancy cells

ABSTRACT Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein, Ad35K++, which is capable of transiently removing CD46 from the cell surface. We also produced a peptide inhibitor of CD59 (rILYd4). In this study, we tested Ad35K++ and rILYd4 in combination with daratumumab and isatuximab in MM cells as well as in cells from two other B-cell malignancies. We showed that Ad35K++ and rILYd4 increased CDC triggered by daratumumab and isatuximab. The combination of both inhibitors had an additive effect in vitro in primary MM cells as well as in vivo in a mouse xenograft model of MM. Daratumumab and isatuximab treatment of MM lines (without Ad35K++ or rILYd4) resulted in the upregulation of CD46/CD59 and/or survival of CD46high/CD59high MM cells that escaped the second round of daratumumab and isatuximab treatment. The escape in the second treatment cycle was prevented by the pretreatment of cells with Ad35K++. Overall, our data demonstrate that Ad35K++ and rILYd4 are efficient co-therapeutics of daratumumab and isatuximab, specifically in multi-cycle treatment regimens, and could be used to improve treatment of multiple myeloma.

Next-Generation Therapies for Multiple Myeloma

Recent therapeutic advances have significantly improved the outcome for patients with multiple myeloma (MM). The backbone of successful standard therapy is the combination of ikaros degraders, glucocorticoids, and proteasome inhibitors that interfere with the integrity of myeloma-specific superenhancers by directly or indirectly targeting enhancer-bound transcription factors and coactivators that control expression of MM dependency genes. T cell engagers and chimeric antigen receptor T cells redirect patients’ own T cells onto defined tumor antigens to kill MM cells. They have induced complete remissions even in end-stage patients. Unfortunately, responses to both conventional therapy and immunotherapy are not durable, and tumor heterogeneity, antigen loss, and lack of T cell fitness lead to therapy resistance and relapse. Novel approaches are under development to target myeloma-specific vulnerabilities, as is the design of multimodality immunological approaches, including and beyond T cells, that simultaneously recognize multiple epitopes to prevent antigen escape and tumor relapse. Expected final online publication date for the Annual Review of Cancer Biology, Volume 8 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Epigenetic Modifiers MMSET and EZH2 Physically Interact and Cooperate to Support Multiple Myeloma Pathophysiology

Multiple myeloma (MM) is a malignant B cell dyscrasia characterized by the accumulation of clonal plasma cells (PC) within the bone marrow (1). Genome sequencing studies have revealed great heterogeneity and genomic instability, a complex mutational landscape and a branching pattern of clonal evolution (2-4). Epigenetic factors are also involved in MM initiation, progression, and occurrence of chemoresistance (5). Among them, EZH2 and MMSET are associated with poor prognosis values and contribute to MM disease. EZH2 is the main catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and deposits H3K27me3 on chromatin (6), whereas MMSET, coded by the gene NSD2, which is the target oncogene of t(4;14) primary translocation, is a histone methyltransferase that methylates H3K36me2 (7). Using proteomics analysis of EZH2-immune precipitates, we identified a constitutive interaction between EZH2 and MMSET independently of t(4;14) translocation and mutational status of EZH2 in MM cells. Furthermore, MAK-683, an allosteric inhibitor of EZH2, disrupted this interaction, but not EPZ-6438, a catalytic inhibitor of EZH2, suggesting that MMSET-EZH2 interaction may be mediated by other PRC2 factors, but is independent of the PRC2 activity. Analysis of EZH2 expression levels and t(4;14) translocation status in 3 independent cohorts of MM patients (CoMMPass (N=674), UAMS-TT2 (N=345), Mtp-Daratumumab (N=51)) identified a worse overall survival of patients with high EZH2 expression combined with t(4;14) chromosomal translocation than patients with only one of these genetic events. We identified 355 differentially expressed genes in this “ EZH2 High t(4;14) +” subgroup compared to other patients. GSEA analysis of up-regulated genes in this subgroup identified a significant enrichment for genes involved in cell cycle, KRAS signaling, Cell-Cell signaling, Hypoxia and Epithelial-Mesenchymal transition. These results indicate that EZH2-MMSET interaction is of clinical interest in t(4;14) MM patients. To better understand the molecular relationship between EZH2 and MMSET, we generated MMSET-depleted clones by CRISPR-Cas9. MMSET depletion did not significantly alter cell proliferation and survival, but reduced the clonogenic capacity of MM cells compared to non-depleted cells. Moreover, after IL-6 or serum starvation, IL-6 and IGF-1 sensitivity was significantly affected in MMSET-depleted clones compared to the parental cell line, indicating that MMSET overexpression confers a cell growth advantage to MM cells under stress conditions and an independence of growth factors produced by bone marrow microenvironment Additionally, MMSET depletion induced changes in PRC2 nuclear localization in MM cells, a decrease of H3K36me3 and a concomitant increase in H3K27me3 levels, which altered the transcriptional program of cells showing, among others, a specific enrichment in genes up-regulated in t(4;14) MM subgroup of patients. Finally, we demonstrated that disrupting EZH2-MMSET interaction with MAK-683 may be of therapeutic interest, since MAK-683 combination with conventional drugs used in MM treatment, including melphalan and Panobinostat or BRD4 inhibitors, synergized to kill MM cells. Taken together, our data identified an “ EZH2 High t(4;14) +” signature in MM patients with a poor outcome who may benefit from combination therapy of MAK-683 with melphalan or epidrugs.

Ex Vivo Mathematical Myeloma Advisor (EMMA) - a Clinical, Molecular, and Phenotypic Platform to Tailor Personalized Therapeutic Strategies for Multiple Myeloma

Multiple myeloma (MM) is a genetically heterogeneous disease, lacking molecular markers for patient stratification and prediction of clinical response. We have created a unique platform for clinical-informed decisions, research advancement, and preclinical compound testing called the “Ex Vivo Mathematical Myeloma Advisor” (EMMA). In this pipeline, MM patients who undergo bone marrow (BM) biopsy and consent to participate in the Total Cancer Care protocol have their clinical data (demographics, therapies received, lab results, clinical history, etc.) abstracted, along with the molecular characterization (FISH/cytogenetics, RNA-Seq, and WES) of their disease. In parallel, fresh BM-derived MM blasts are combined with stromal cells in a collagen-based matrix containing patient plasma and used in an ex vivo drug screening assay. The cells are seeded into 384 or 1536-well plates and exposed to serial dilutions of up to 127 single agents and combinations. The entire setup is imaged in bright field every 30 minutes for a duration of 6 days. Image analysis is used to convert membrane integrity into survival curves, and AUC and LD50 values are estimated. The pattern of these curves facilitates the identification of resistant subclones. This data is further combined with augmented synergy models that are parameterized with PK/PD data for each drug. This integration permits the simulation of “in silico clinical trials”, which are compiled into reports that assist clinicians in selecting the best treatment for their patients. In addition, the immune population of the BM aspirate is characterized by flow cytometry, and soluble factors in the patient's BM plasma are quantified by ELISA. The integration of clinical, molecular, and phenotypic data with image analysis and machine learning tools presents a unique opportunity for finding molecular signatures of drug response (Fig. 1). For instance, our pipeline can be utilized to identify clinical factors (such as ethnicity, age, etc.) and molecular features (mutations, cytogenetic abnormalities, enriched transcription factors, differentially expressed biological pathways, etc.) associated with drug resistance/sensitivity. Additionally, it enables the determination of the mechanisms of action of new compounds and the prediction of potential synergistic/antagonistic drug combinations. The longitudinal acquisition of data in our non-destructive ex vivo drug screen assay facilitates the exploration of drug interaction dynamics over time, enabling the pinpointing of the moment when synergism occurs at maximum intensity. This, in turn, allows us to propose optimized temporal-based therapeutic regimens for clinical use. Up to this point, our pipeline has molecularly characterized nearly 1,000 MM samples, and EMMA has been applied to over 600 samples, forming one of the world's most extensive and comprehensively characterized MM cohorts. While EMMA has proven effective in quantifying the activity of monoclonal antibodies, ongoing efforts are focusing on incorporating individual immune cell components to assess ex vivo activity in the context of evolving cellular immunotherapies. As a proof of principle, we demonstrate the system's capability to measure the effect of daratumumab (CD38+ antibody) on autologous macrophage-mediated MM cell death, thereby distinguishing it from apoptosis caused by direct antibody binding (Fig. 2). Additionally, we evaluate the influence of teclistamab (T-cell engager CD3/BCMA bispecific antibody) on CD8+ T cell activity against autologous MM cells, as well as the efficacy of CAR-T cells in killing MM cells. Through the testing of standard-of-care drugs alongside immune agents, our pipeline has identified the combination of bortezomib + daratumumab as a synergistic approach with significant translational potential, warranting further investigation. In summary, our state-of-the-art platform facilitate drug discovery and unravel the intricate biological phenomena underlying drug interactions in MM. The advantages offered by our system are threefold: i) it enables pharmaceutical companies to test new compounds in a bona fide, patient-derived model; ii) it assists clinicians in selecting the best therapy for their patients, and iii) it helps prevent cancer patients from receiving non-suitable therapies. This approach can also be promptly applied to other liquid cancers, such as leukemia and lymphoma.

Higher CD56 Expression on Multiple Myeloma Cells Increases CD38 Expression, Reduces Intracellular NAD+ Levels, and Enhances the Efficacy of Daratumumab-Based Treatment Strategies

NCAM1 (Neural Cell Adhesion Molecule 1), also known as CD56, is a member of the immunoglobulin superfamily and is deregulated in many tumors. In multiple myeloma (MM), high levels of CD56 are observed on MM cells in 70% of patients. This molecule has several functions including tumor growth, adhesion and response to therapy, as suggested by several studies. However, an extensive analysis of the specific landscape underlying its presence and its role as a prognostic biomarker is lacking, especially in MM. Here, we evaluated CD56 surface levels on tumor cells collected from 136 newly diagnosed MM patients (Policlinic San Martino Hospital, Genoa, Italy) and found that low CD56 expression significantly correlated with extramedullary disease (EMD) (p = .016) while high CD56 levels were associated with a lower risk of disease progression or death (p = .003). When evaluating the surface expression of MM cells, we surprisingly found a positive correlation between CD56 and CD38 levels (p < .0001), which was replicated in a panel of MM cell lines (p = .02). To gain insight into the biological significance of this, we challenged a panel of MM cell lines with Daratumumab (DARA), a standard-of-care CD38 antibody that kills MM cells through multiple mechanisms including antibody-dependent cellular cytotoxicity (ADCC). Given the correlation between the expression of CD56 and CD38, we hypothesized that higher CD56 expression would lead to enhanced DARA-mediated ADCC. We performed standard 3-hour ADCC assays using NK cells from healthy donors as effector cells and CD56-positive or -negative cell lines. Myeloma cells were stained with calcein-AM and co-cultured with NK cells at indicated effector:target (E:T) ratios. The assay was assessed in the presence or absence of 1μg/mL Daratumumab. We found that MM cell lines with high expression of CD56 were more sensitive to DARA-mediated NK cell killing (p = .004 at ratio 1:1 E:T). Consistently, MM patients with higher levels of CD56 (N = 8) had better overall response rates to DARA-based therapies compared to patients with lower levels (N = 9). CD56 and CD38 are not known to interact, so this correlation may be mediated by the NAD+ biosynthesis pathway. CD38 is a critical enzyme for breaking down NAD + in cells. We found that ectopic expression of CD56 led to lower intracellular NAD + levels (MM1S overexpressing CD56 p = .0875; RPMI 8226 overexpressing CD56 p = .0003) and increased CD38 enzymatic activity (MM1S overexpressing CD56 p = .0035; RPMI 8226 overexpressing CD56 p = .0478), as measured by a cyclase activity assay. As a result, CD56 surface levels affected MM cells' sensitivity to NAD +-lowering agents, including next-generation NAMPT inhibitors. Indeed, MTS assays confirm that CD56-overexpressing MM cells were more vulnerable to these small molecules, whereas CD56 silencing made cells more resistant. In conclusion, our data suggest CD56 levels as an important determinant of sensitivity to DARA-based therapies. Moreover, our results suggest the next-generation NAMPT inhibitors as an additional therapeutic strategy for CD56-expressing MM patients.

Iberdomide Enhances Dara Mediated Cytotoxicity through Upregulation of CDC Activity and Elevated NK Cell Mediated ADCC

Background: Multiple Myeloma (MM) is an incurable hematological malignancy with current treatments comprising combination regimens, typically featuring immunomodulatory agents (IMiDs) due to their potent anti-tumor activity and immunomodulatory effects. Clinical studies utilizing the combination of IMiD agents and daratumumab (DARA), an anti-CD38 monoclonal antibody, have shown improved patient outcomes for MM. However, patients ultimately relapse demonstrating the need for novel therapies. Iberdomide (IBER) is a novel cereblon E3 ligase modulator. It promotes faster and deeper degradation of the substrate targets compared to IMiDs, resulting in enhanced anti-proliferative, pro-apoptotic and immune stimulatory activity. In early clinical studies, IBER has been shown to overcome resistance to IMiDs and in the ongoing Phase 3 EXCALIBER study (NCT04975997) is being explored for treatment of newly diagnosed (ND) and relapsed/refractory (RR) MM in combination with DARA and dexamethasone. Here, we investigate the potential mechanisms of synergy between IBER and DARA in preclinical models. Methods: Complement dependent cytotoxicity (CDC) of DARA was assessed with 15% human serum for human complement components and assays were performed by titrating either single agent or combinations in a tritiated thymidine assay. Antibody dependent cellular cytotoxicity (ADCC) was performed by co-culturing MM cells with varying expression levels of CD38 and effector cells and assessing target cell viability with annexin-v/topro-3 flow. A humanized PBMC adoptive transfer mouse model, utilizing human PBMC engraftment 14 days prior to implantation, was used to assess in vivo activity of IBER, DARA and IBER + DARA. Results: In CDC assays, we observed variable sensitivity of MM cells to complement-specific CDC in the presence of DARA (0.1-1 µg/ml), regardless of cell surface CD38 expression. Notably, IBER treatment resulted in upregulation of CD38 surface expression in 60% of MM cell lines tested. LP1 and MOLP8 displayed susceptibility to DARA-mediated CDC. DARA showed a dose-dependent increase in CDC activity in both cell lines. Notably, IBER+DARA also showed ~35% more synergistic CDC activity in both cell lines, than the single agents. In ADCC assays, we observed pretreatment of PBMC with IBER (vs. DMSO) significantly increased DARA mediated ADCC in H929 and LP-1 cells. To confirm that this effect was directed by NK cells, we cultured NK cells for 24 hr in supernatants from stimulated PBMC in presence of IBER prior to co-culture with MM cells. We noted that IBER-treated PBMC supernatant significantly enhanced the NK ADCC activity of DARA suggesting that IBER enhancement of cytokine secretion from PBMC increases NK cell mediated ADCC. Orthogonally, we pre-treated MM cells with IBER or DMSO prior to co-culture in ADCC assay. Notably, IBER treatment of MM cells, without treating immune cells, also increased DARA-mediated ADCC, indicating that IBER directly increases sensitivity to DARA-directed ADCC. To investigate the MOA of IBER-DARA synergy in ADCC, we analyzed gene expression and cell surface changes of a panel of NK-specific receptor ligands on MM cells following treatment with IBER. We observed IBER-induced upregulation of MICA mRNA levels (~2x) and surface expression (~40%) in MM target cells. We used RNAi to knock down Ikaros, an IBER substrate, which resulted in increased MICA expression (~3x) in MM cells. Furthermore, we observed that RNAi knock down of MICA in MM cells resulted in a significant decrease (60%, p<0.0001) in MM cell killing by NK cells, suggesting IBER-enhanced sensitivity of MM cells to NK-cell killing was in part, due to the upregulation of MICA through degradation of Ikaros. Lastly, we examined the anti-MM effects of IBER in a humanized xenograft model with a lenalidomide resistant cell line. Longitudinal analysis identified persistence of human PBMC engraftment and activation of immune subsets (NK cells and T-cells) that were increased upon IBER treatments, consistent with clinical observations. The combination of IBER/DARA resulted in ~22% tumor growth inhibition (TGI) in comparison to single agents, DARA and IBER and ~37% TGI in comparison to vehicle. Conclusion: Taken together, these data demonstrate that IBER and DARA work synergistically in preclinical models primarily through enhancement of both ADCC and CDC. This combination has the potential to improve outcomes in MM.

Bivalent BCMA Binding and Low Affinity CD3 T-Cell Engagement By Abbv-383 Drives Sustained Activation with Reduced T-Cell Exhaustion in Preclinical Models of Multiple Myeloma

Introduction: ABBV-383 is an IgG4-based CD3-bispecific T-cell engager (TCE) that has shown promising efficacy and a favorable safety profile in heavily pre-treated patients with relapsed/refractory multiple myeloma (MM). ABBV-383 possesses a unique combination of a low-affinity CD3δε antibody coupled to a bivalent, high-affinity BCMA engager arm in a 2+1 format. Prior pre-clinical studies demonstrated that ABBV-383 induces potent redirected T-cell killing of MM tumors with reduced cytokine release. Here, we provide additional evidence that bivalent BCMA binding by ABBV-383 reduces the negative impact of soluble BCMA and low affinity CD3 T-cell engagement drives sustained T-cell activation with reduced immune exhaustion. Methods: Structural variants of ABBV-383 were created, including 1) the bivalent BCMA targeting antibody of ABBV-383 replaced by proximal and distal monovalent VH targeting molecules, and 2) BCMA antibody structures coupled to high-and low affinity CD3 variants ( Figure). For functional testing, ABBV-383 and the structural variants were added to PBMCs or T-cells from healthy donors to serve as effector cells in co-culture with BCMA-positive MM tumor cell lines. Tumor cell killing and T-cell activation were determined by flow cytometry, and cytokine production was assessed using Luminex instrumentation. Recombinant soluble BCMA (sBCMA) was utilized in the experimental models to evaluate the extent of BCMA TCE suppression. To define the relationship between CD3-affinity and T-cell exhaustion, multi-dimensional flow cytometry was performed in long-term, 28-day co-cultures with irradiated tumor cells replated every 7 days. Results: T-cell binding was shifted by >100x with substitution of the high affinity CD3 antibody compared to ABBV-383. While all antibody variants tested induced T-cell specific MM cell killing in a BCMA-dependent manner, the negative impact of soluble BCMA on tumor killing was reduced with ABBV-383 due to its bivalent interaction with tumor cells compared to the monovalent TCE formats. In addition, the low CD3 binding affinity of ABBV-383 lowered T-cell activation antigen expression and reduced inflammatory cytokine production but resulted in similar maximal cytotoxicity of tumor cells in co-cultures of healthy and MM patient samples compared to high-affinity CD3 targeting. While maximal tumor cytotoxicity was similar, ABBV-383 was found to significantly delay the induction of a critical exhaustion-inducing transcription factor, TOX, in long-term assays. T-cell population changes were observed as early as 3 days with decreased effector memory cells (CD45RA-CD62L-) and increased naïve T-cell populations (CD62L+CD45RA+) compared to high-affinity CD3 targeting. On day 21, the high-affinity CD3 x bivalent BCMA molecule resulted in 85% of the CD8+ T-cell population expressing the transcription factor TOX compared to only 15% for ABBV-383 co-cultures. TOX induces several gene families with chromatin-modulating capacity that drive the distinct transcriptional trajectory leading to T-cell dysfunction during chronic stimulation. Simultaneous expression of multiple checkpoint inhibitory proteins is indicative of T-cell exhaustion. Triple positive CD8+ T-cells expressing LAG3, PD-1 and CTLA4 were significantly reduced by ABBV-383 versus the high-affinity CD3 variant ( Figure). Conclusions: High avidity BCMA binding coupled to low affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383 in MM. Here, we show that the presence of bivalent-BCMA reduced the negative impact of soluble BCMA, reduced inflammatory cytokine production, and dampened the emergence of TOX+ CD8+ T-cells indicating that ABBV-383 structure maximizes its clinical potential as a safe and effective MM therapy.

Pharmacologic targeting of the p62 ZZ domain enhances both anti-tumor and bone-anabolic effects of bortezomib in multiple myeloma.

Multiple myeloma (MM) is a malignancy of plasma cells whose antibody secretion creates proteotoxic stress relieved by the N-end rule pathway, a proteolytic system that degrades Narginylated proteins in the proteasome. When the proteasome is inhibited, protein cargo is alternatively targeted for autophagic degradation by binding to the ZZ-domain of p62/sequestosome-1. Here, we demonstrate that XRK3F2, a selective ligand for the ZZ-domain, dramatically improved two major responses to the proteasome inhibitor bortezomib by increasing: 1) killing of human MM cells by stimulating both bortezomib mediated apoptosis and necroptosis, a process regulated by p62; and 2) preservation of bone mass by stimulating osteoblasts differentiation and inhibiting osteoclastic bone destruction. Co-administration of bortezomib and XRK3F2 inhibited both branches of the bimodal N-end rule pathway exhibited synergistic anti-MM effects on MM cell lines and CD138+ cells from MM patients, and prevented stromal-mediated MM cell survival. In mice with established human MM, coadministration of bortezomib and XRK3F2 decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inducing new bone formation more effectively than either single agent alone. The results suggest that p62-ZZ ligands enhance the anti-MM efficacy of proteasome inhibitors and can reduce MM morbidity and mortality by improving bone health.

Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells

We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM–3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15–23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.

Crosstalk between glucocorticoid and mineralocorticoid receptors boosts glucocorticoid-induced killing of multiple myeloma cells

The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here, we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that plays a role in improved myeloma cell killing. We show that the GR agonist dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR–MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR–MR heterodimerization and completely abolishes Dex-induced MR–MR homodimerization, while leaving GR–GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR–MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.

CAMMA 2: A phase I/II trial evaluating the efficacy and safety of cevostamab in patients with relapsed/refractory multiple myeloma (RRMM) who have triple-class refractory disease and have received a prior anti-B-cell maturation antigen (BCMA) agent.

TPS8064 Background: Multiple myeloma (MM) remains an incurable disease. Combinations of established agents, including proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and anti-CD38 antibodies, are used in all lines of therapy, but re-exposure to previously received classes of therapy is associated with decreasing response rates and duration of response (Moreau et al. 2021). BCMA-targeted agents have recently been approved for the treatment of patients with RRMM whose disease has been exposed or become refractory to PIs, IMIDs and anti-CD38 antibodies (triple-class exposed or refractory MM). Although effective, the majority of patients who respond to anti-BCMA agents eventually relapse. Proven salvage therapies for these patients are currently lacking and represent a new unmet medical need. Cevostamab is an immunoglobulin G1-based T-cell-engaging bispecific antibody that targets Fc receptor-homolog 5 (FcRH5) on myeloma cells and CD3 on T cells. Dual binding results in T-cell activation and potent killing of MM cells. In an ongoing Phase I study (GO39775; NCT03275103), cevostamab monotherapy showed promising activity in heavily pre-treated patients with RRMM, including those with prior exposure to anti-BCMA agents (Trudel et al. 2021). Methods: CAMMA 2 (CO43476; NCT05535244) is an open-label, multicenter Phase I/II trial evaluating cevostamab monotherapy in patients with RRMM who are triple-class refractory and have previously received an anti-BCMA agent. Prior anti-BCMA antibody-drug conjugates or chimeric antigen receptor (CAR) T cells are permitted in Cohorts A1 and B1, while prior anti-BCMA bispecific antibodies are allowed in Cohorts A2 and B2. In A1 and A2, cevostamab is administered by intravenous infusion and is initiated with step dosing (0.3mg on Cycle [C] 1 Day [D] 1 and 3.3mg on C1D2, D3, or D4 depending on the emergence and resolution of cytokine release syndrome [CRS] after the initial administration), with the target dose of 160mg given on C1D8 and on D1 of each subsequent 21-day cycle. Patients are hospitalized for the C1 administrations only and treatment is continued until disease progression or unacceptable toxicity occurs. CRS is managed per protocol based on the clinical presentation and may involve corticosteroid and tocilizumab treatment. Primary objectives are evaluation of efficacy (primary endpoint: objective response rate by investigator assessment using IMWG criteria) and safety. Secondary objectives include assessment of quality of life, pharmacokinetics, pharmacodynamics, and immunogenicity. Cohorts B1 and B2 will evaluate the efficacy and safety of cevostamab at the recommended Phase II dose in this setting. As of February 2023, enrolment into Cohorts A1 and A2 is ongoing. Clinical trial information: NCT05535244 .

NK Cell Phenotype Is Associated With Response and Resistance to Daratumumab in Relapsed/Refractory Multiple Myeloma.

The CD38-targeting antibody daratumumab has marked activity in multiple myeloma (MM). Natural killer (NK) cells play an important role during daratumumab therapy by mediating antibody-dependent cellular cytotoxicity via their FcγRIII receptor (CD16), but they are also rapidly decreased following initiation of daratumumab treatment. We characterized the NK cell phenotype at baseline and during daratumumab monotherapy by flow cytometry and cytometry by time of flight to assess its impact on response and development of resistance (DARA-ATRA study; NCT02751255). At baseline, nonresponding patients had a significantly lower proportion of CD16+ and granzyme B+ NK cells, and higher frequency of TIM-3+ and HLA-DR+ NK cells, consistent with a more activated/exhausted phenotype. These NK cell characteristics were also predictive of inferior progression-free survival and overall survival. Upon initiation of daratumumab treatment, NK cells were rapidly depleted. Persisting NK cells exhibited an activated and exhausted phenotype with reduced expression of CD16 and granzyme B, and increased expression of TIM-3 and HLA-DR. We observed that addition of healthy donor-derived purified NK cells to BM samples from patients with either primary or acquired daratumumab-resistance improved daratumumab-mediated MM cell killing. In conclusion, NK cell dysfunction plays a role in primary and acquired daratumumab resistance. This study supports the clinical evaluation of daratumumab combined with adoptive transfer of NK cells.

Abstract LB209: An ADC composed of daratumumab and lenalidomide is extremely powerful in killing multiple myeloma cells

Abstract Introduction: Multiple myeloma (MM) is a neoplastic malignancy characterized by the abnormal proliferation of plasma cells with excessive antibody production in the bone marrow. It accounts for approximately 1.8% of all new cancer cases and 2.1% of cancer deaths in the United States and occurs most frequently among older adults (age > 75). While there are a number of treatments with high response rates, MM relapses frequently within a few years and is considered incurable. Herein, experimental studies and results of a new therapeutic agent under development, TE-1146, are reported. TE-1146 is an antibody drug conjugate (ADC) composed of two therapeutic agents already in use in MM, namely, an anti-CD38 antibody, daratumumab (Dara), and lenalidomide (Lena). Method: TE-1146 is designed by employing “HIDAR” technology platform, which enables the preparation of homogeneous ADCs with high DAR (drug to antibody ratio). In the TE-1146 molecules, the Fab is replaced by scFv and a cysteine-containing Zn2+-binding motif is engineered at the C-termini of the H chains. Lena molecules are assembled into drug bundles containing 3 Lena molecules and a maleimide group. Two drug bundles are conjugated site-specifically to the Zn2+-activated SH groups of the reconfigured antibody molecule, creating an ADC with DAR of 6. TE-1146 was investigated for cytolytic effects against human MM cell lines H929 in cell cultures in vitro and transplanted H929 tumor in NOD-SCID mice in vivo, in comparison with Dara, Lena, and their combination. Results: In human plasma, TE-1146 has stability comparable with Dara, and the Lena molecules in the drug bundles remain conjugated. In cultures of H929 cells, TE-1146 caused the lysis of H929 cells at least 100 times more effectively than Dara, Lena, and Dara/Lena combination, based on IC50 comparison. It was shown that H929 cells internalized and degraded TE-1146 and freed Lena. In NOD-SCID mice, the subcutaneously transplanted H929 cells were allowed to grow into solid tumors in 14 days, and TE-1146 and other agents for comparison were administered intraperitoneally. It was found that one single dose of TE-1146 at 20 nmol/kg (conjugated with Lena at 120 nmol/kg) could retard the growth of the transplanted tumor and ultimately eliminate the tumor over 28-42 days, while the combination of one dose Dara at 20-80 nmol/kg and Lena of 46 μmol/kg given daily could not eliminate the tumor. The amount of Lena used in the combination treatment over a 28-day course is 10,700 times that of Lena in TE-1146. Conclusion: Lenalidomide is extremely powerful in killing multiple myeloma cells if brought into the MM cells. It is estimated that a very minute amount, probably less than 0.01%, of intraperitoneally injected lenalidomide gets into the transplanted MM tumor in the mouse model. TE-1146 may be a more effective and less toxic drug than Daratumumab/Lenalidomide combination in treating patients with hard-to-treat MM. Citation Format: Yueh-Hsiang Yu, Ming-Yu Hsieh, Pei-Wen Wu, Hui-Ju Lee, Pei-Hsuan Lin, Carmay Lim, Hsing-Mao Chu, Tse Wen Chang. An ADC composed of daratumumab and lenalidomide is extremely powerful in killing multiple myeloma cells [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 LB209.

Antibody Surface Profiling Identifies Glycoforms in Multiple Myeloma as Targets for Immunotherapy: From Antibody Derivatives to Mimetic Peptides for Killing Tumor Cells

Despite therapeutic advances in recent years, there are still unmet medical needs for patients with multiple myeloma (MM). Hence, new therapeutic strategies are needed. Using phage display for screening a large repertoire of single chain variable fragments (scFvs), we isolated several candidates that recognize a heavily sulfated MM-specific glycoform of the surface antigen syndecan-1 (CD138). One of the engineered scFv-Fc antibodies, named MM1, activated NK cells and induced antibody-dependent cellular cytotoxicity against MM cells. Analysis of the binding specificity by competitive binding assays with various glycan ligands identified N-sulfation of glucosamine units as essential for binding. Additionally, site-directed mutagenesis revealed that the amino acids arginine and histidine in the complementarily determining regions (CDRs) 2 and 3 of the heavy chain are important for binding. Based on this observation, a heavy-chain antibody, known as a nanobody, and a peptide mimicking the CDR loop sequences were designed. Both variants exhibited high affinity and specificity to MM cells as compared to blood lymphocytes. Specific killing of MM cells was achieved by conjugating the CDR2/3 mimic peptide to a pro-apoptotic peptide (KLAKLAK)2. In a co-culture model, the fusion peptide killed MM cells, while leaving normal peripheral blood mononuclear cells unaffected. Collectively, the development of antibodies and peptides that detect tumor-specific glycoforms of therapeutic targets holds promise for improving targeted therapies and tumor imaging.

Anti-BCMA surface engineered biomimetic photothermal nanomissile enhances multiple myeloma cell apoptosis and overcomes the disturbance of NF-κB signaling in vivo

Conventional chemotherapy for multiple myeloma (MM) faces the challenges of a low complete remission rate and transformation to recurrence/refractory. The current MM first-line clinical drug Bortezomib (BTZ) faces the problem of enhanced tolerance and nonnegligible side effects. B cell maturation antigen (BCMA), for its important engagement in tumor signaling pathways and novel therapy technologies such as Chimeric antigen receptor T-Cell immunotherapy (CAR-T) and Antibody Drug Conjugate (ADC), has been identified as an ideal target and attracted attention in anti-MM therapy. Emerging nanotechnology provided feasible methods for drug delivery and new therapeutic strategies such as photothermal therapy (PTT). Herein, we developed a BCMA-Targeting biomimetic photothermal nanomissile BTZ@BPQDs@EM @anti-BCMA (BBE@anti-BCMA) by integration of BTZ, black phosphorus quantum dots (BPQDs), Erythrocyte membrane (EM) and BCMA antibody (anti-BCMA). We hypothesized that this engineered nanomissile could attack tumor cells in triple ways and achieve effective treatment of MM. Consequently, the intrinsic biomimetic nature of EM and the active targeting property of anti-BCMA enhanced the accumulation of therapeutic agents in the tumor site. Besides, owing to the decrease in BCMA abundance, the potential apoptosis-inducing ability was revealed. With the support of BPQDs' photothermal effect, Cleaved-Caspase-3 and Bax signal increased significantly, and the expression of Bcl-2 was inhibited. Furthermore, the synergistic photothermal/chemo therapy can effectively inhibit tumor growth and reverse the disorder of NF-κB in vivo. Importantly, this biomimetic nanodrug delivery system and antibody induced synergistic therapeutic strategy efficiently killed MM cells with ignorable systemic toxicity, which is a promising method for the future anticancer treatment of hematological malignancies in clinics.

Inhibition of PI3K Class IA Kinases Using GDC-0941 Overcomes Cytoprotection of Multiple Myeloma Cells in the Osteoclastic Bone Marrow Microenvironment Enhancing the Efficacy of Current Clinical Therapeutics.

Osteoclasts contribute to bone marrow (BM)-mediated drug resistance in multiple myeloma (MM) by providing cytoprotective cues. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity in MM. Although targeting osteoclast function is critical to improve MM therapies, pre-clinical studies rarely consider overcoming osteoclast-mediated cytoprotection within the selection criteria of drug candidates. We have performed a drug screening and identified PI3K as a key regulator of a signalling node associated with resistance to dexamethasone lenalidomide, pomalidomide, and bortezomib mediated by osteoclasts and BM fibroblastic stromal cells, which was blocked by the pan-PI3K Class IA inhibitor GDC-0941. Additionally, GDC-0941 repressed the maturation of osteoclasts derived from MM patients and disrupted the organisation of the F-actin cytoskeleton in sealing zones required for bone degradation, correlating with decreased bone resorption by osteoclasts. In vivo, GDC-0941 improved the efficacy of dexamethasone against MM in the syngeneic GFP-5T33/C57-Rawji mouse model. Taken together, our results indicate that GDC-0941 in combination with currently used therapeutic agents could effectively kill MM cells in the presence of the cytoprotective BM microenvironment while inhibiting bone resorption by osteoclasts. These data support investigating GDC-0941 in combination with currently used therapeutic drugs for MM patients with active bone disease.

Modakafusp Alfa (TAK-573), a Novel CD38-Targeting Attenuated Interferon-Alpha Immunocytokine, Kills MM Cells Via NK Cell Activation

Background Despite recent success of monoclonal antibodies (mAb) and T cell redirecting therapies, relapsed/refractory (RR) multiple myeloma (MM) patients remain in urgent need of new therapies with distinct mechanisms of action (MoA). Modakafusp alfa, previously known as TAK-573, is a novel immune-targeted attenuated cytokine specifically designed to deliver interferon-alpha to CD38-expressing cells via a CD38-targeting IgG4 mAb. Preliminary results of a first in human phase 1 trial with modakafusp alfa monotherapy (1.5mg/kg, every four weeks) in RRMM patients (90% anti-CD38 mAb refractory; 37% anti-BCMA agent exposed) indicate a promising 40% overall response with manageable toxicity (Kaufman et al. EHA 2022 abstract S181). Prior studies suggest a unique and multimodal MoA of modakafusp alfa, including anti-proliferative effects on tumor cells as well as stimulation of CD38-positive immune effector cells. Towards a thorough understanding of these immunomodulatory effects, specifically regarding the activation of innate immunity, we here studied the effects of modakafusp alfa on the anti-MM activity of NK cells, which highly express CD38. Aims We assessed the ex vivo activity of modakafusp alfa on NK cell activation, degranulation, and antibody-independent cytotoxicity. In addition, we investigated the impact of modakafusp alfa on NK cell-mediated, antibody-dependent cellular cytotoxicity (ADCC) against MM cells using daratumumab (anti-CD38) or elotuzumab (anti-SLAMF7). Methods NK cell activation was explored by flow cytometry after 24-hour incubation of peripheral blood mononuclear cells (PBMC) from healthy donors with modakafusp alfa or negative/specificity controls. These pre-incubated PBMCs were also used to examine NK cell degranulation after subsequent 4 hour stimulation with K562 or PMA/Ionomycin, or in a 16-hour cytotoxicity assay against K562. In addition, modakafusp alfa pre-incubated PBMCs or enriched NK cells were assessed for antibody-independent cytotoxicity or daratumumab/elotuzumab-mediated ADCC in 24-48 hour assays against MM cell lines RPMI-8226, MM.1S or UM9. Alternatively, modakafusp alfa was directly added to the assays without pre-incubation steps. Results We found that incubation of PBMCs with modakafusp alfa for 24 hours resulted in upregulation of activation markers CD38 and CD69 on the surface of NK cells. In addition, modakafusp alfa pre-incubation significantly improved the degranulation of NK cells after 4-hour stimulation with K562 or PMA/Ionomycin. Importantly, overnight pre-incubation of PBMCs or enriched NK cells with modakafusp alfa showed enhanced antibody-independent cytotoxicity against K562 tumor cells and MM cell lines RPMI-8226, MM.1S or UM9. Consequently, this also resulted in augmented killing of MM cells in the presence of daratumumab or elotuzumab. We observed similar effects when modakafusp alfa was directly added to the cytotoxicity assays without pre-incubation of immune effector cells. Conclusions Modakafusp alfa rapidly activates NK cells, and improves their degranulation and anti-MM effectivity. This important MoA also results in improved efficacy of daratumumab or elotuzumab in short-term cytotoxicity assays. Our findings warrant clinical investigation of combining modakafusp alfa with NK cell-exploiting immunotherapies.

HSV1716 Infects Multiple Myeloma Cells and Leads to CD138 Downregulation

Multiple myeloma (MM) is a plasma cell malignancy that accounts for approximately 10% of all hematological cancers. MM is classified as incurable due to a high relapse rate after the initial therapy and failure to achieve long-term minimal residual disease (MRD). Therefore, new therapies and combinations are needed. Oncolytic viruses (OVs) are genetically modified viruses that preferentially infect and lyse malignantly transformed cells. HSV1716 is a replication-restricted herpes-simplex virus type I (HSV-1)-based OV (HSV-OV) which has been evaluated in several clinical trials. HSV-OVs have been shown to infect and kill MM cell lines and primary malignant plasma cells. Furthermore, HSV-OV mediated target cell killing was demonstrated to be enhanced by natural killer (NK) cells, an immune cell subset that naturally recognizes malignantly transformed or virally infected cells. The underlying mechanisms behind HSV-OV-enhanced NK-tumor cell killing, however, have not yet been elucidated. Here, we aim to monitor phenotypic changes of MM cells upon HSV1716 infection and thus explore the basis for NK cell recognition of HSV-OV infected target MM cells. The MM cell lines RPMI8226 and MM1S were infected with HSV1716 at various multiplicities of infection (MOIs) over a three-day timecourse. Target cell death and expression of viral glycoproteins were measured at different timepoints by flow cytometry. HSV1716 caused oncolysis of 60% of RPMI8226, and 70% of MM1S cells 48h post-infection (p.i.) at an MOI of 0.5. Under these conditions, the expression of viral glycoprotein E on live cells reached 74% on RPMI8226 cells and 37% on MM1S cells. Primary NK (pNK) cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and co-cultured with infected and uninfected MM cells for 4 hours. CD107a as a marker for NK cell degranulation, as well as the release of IFNγ and TNF were measured on CD3-CD56+ pNK cells by flow cytometry. CD107a expression increased from 15.6% (+/- 0.9%) to 56.2% (+/- 2.4%) following infection of RPMI8226 cells and from 15.6% (+/- 3.4%) to 59.2% (+/- 4.5%) following infection of MM1S cells. Similarly, the expression of IFNγ and TNF increased by four- and three-fold, respectively, as compared to uninfected target cells. To monitor changes in the expression of NK cell ligands on infected MM cells, a 16-color flow cytometry panel was designed. RPMI8226 and MM1S cells were infected at different MOIs, and ligand expression was analyzed at 24h, 48h, and 72h p.i.. Most significantly, an early and progressive downregulation of CD138 expression from 96% on uninfected cells to 79 % (24h MOI 0.5), 70% (48h MOI 0.5), and 22% (72h MOI 0.5) on live RPMI8226 cells and from 99% on uninfected cells to 82% (24h MOI 0.5), 65% (48h MOI 0.5) and 23% (72h MOI 0.5) on live MM1S cells was observed. Of note, the expression of CD38 remained stable at each of these timepoints. While downregulation of HLA class I molecules was observed, the expression of other NK cells ligands on RPMI8226 or MM1S cells did not notably differ between infected and uninfected cells. Together, our data confirm that HSV-OV enhances NK cell-mediated killing of MM cells. While expression of ligands for activating NK cell receptors was not altered on infected versus uninfected RPMI8226 and MM1S cells, a strong downregulation of CD138 with maintained CD38 expression was observed upon HSV1716 infection. The implementation of oncolytic virotherapy as an immunotherapeutic platform for cancer treatment adds a new and promising therapeutic tool for combination therapies to the treatment options of MM.