Lysis Of Multiple Myeloma Cells Research Articles

Preclinical evaluation of the CD38-targeting engineered toxin body MT-0169 against multiple myeloma.

Despite significant progress in the treatment of multiple myeloma (MM), relapsed/refractory patients urgently require more effective therapies. We here describe the discovery, mechanism of action, and preclinical anti-MM activity of engineered toxin body MT-0169, a next-generation immunotoxin comprising a CD38-specific antibody fragment linked to a de-immunized Shiga-like toxin A subunit (SLTA) payload. We show that specific binding of MT-0169 to CD38 on MM cell lines triggers rapid internalization of SLTA, causing cell death via irreversible ribosome inhibition, protein synthesis blockade, and caspase 3/7 activation. In co-culture experiments, bone marrow mesenchymal stromal cells did not induce drug resistance against MT-0169. In the preclinical setting, MT-0169 effectively lysed primary MM cells from newly diagnosed and heavily pretreated MM patients, including those refractory to daratumumab, with minimal toxicity against nonmalignant hematopoietic cells. MM cell lysis showed a significant correlation with their CD38 expression levels but not with cytogenetic risk, tumor load, or number of prior lines of therapy. Finally, MT-0169 showed efficient in vivo anti-MM activity in various mouse xenograft models, including one in which MM cells are grown in a humanized bone marrow-like niche. These findings support clinical investigation of MT-0169 in relapsed/refractory MM patients, including those refractory to CD38-targeting immunotherapies.

Evaluation of Drug-Drug Interaction Potential of Talquetamab, a T-Cell-Redirecting GPRC5D×CD3 Bispecific Antibody, as a Result of Cytokine Release Syndrome in Patients with Relapsed/Refractory Multiple Myeloma in MonumenTAL-1, Using a Physiologically Based Pharmacokinetic Model.

Cytokine release syndrome, commonly associated with T-cell immunotherapies, was observed with talquetamab, a T-cell-redirecting bispecific antibody, in the phase I/II MonumenTAL-1 study, leading to elevated interleukin (IL)-6, which can suppress cytochrome P450 (CYP) enzyme activity. We aimed to evaluate the potential impact of elevated IL-6 on the exposure of co-administered CYP450 substrates for two scenarios: (1) the observed median IL-6 profile and (2) a profile with the highest IL-6 maximum concentration following talquetamab treatment. A physiologically based pharmacokinetic model was developed based on the literature and simulations performed using observed IL-6 profiles from patients in MonumenTAL-1 who received the subcutaneous recommended phase 2 doses (RP2Ds) of talquetamab: 0.4 mg/kg weekly (QW) and 0.8 mg/kg every other week (Q2W). Median IL-6 maximum concentration was 18.4 and 7.1 pg/mL, and maximum IL-6 maximum concentration was 213 and 3503 pg/mL for talquetamab QW and Q2W RP2Ds, respectively. For the median IL-6 profile, no interaction between IL-6 and studied CYP substrates was predicted at either RP2D. The maximum IL-6 profile predicted weak-to-moderate impact on exposure of CYP2C19, CYP3A4, and CYP3A5 substrates and minimal impact on exposure of CYP1A2 substrates at both RP2Ds. Impact on exposure of CYP2C9 substrates was predicted as minimal at QW and minimal-to-weak at Q2W RP2Ds. Time to return to 20% difference from baseline enzymatic activity was predicted as 7 and 9 days after start of cycle 1 for QW and Q2W RP2Ds, respectively. These modeling results suggest that IL-6 release due to talquetamab-induced cytokine release syndrome has limited impact on potential drug-drug interactions, with the highest likelihood of impact occurring from initiation of talquetamab step-up dosing up to 7 (QW) or 9 (Q2W) days after first treatment dose in cycle 1 and during and after cytokine release syndrome. Multiple myeloma can be treated with immunotherapies such as the bispecific antibody, talquetamab, which binds the novel antigen G protein-coupled receptor family C group 5 member D on multiple myeloma cells and CD3 on T cells and induces T-cell-mediated lysis of multiple myeloma cells. Following talquetamab treatment, many patients experience cytokine release syndrome, an inflammatory immune response where levels of proinflammatory cytokines, including interleukin (IL)-6, are increased. Interleukin-6 can suppress the activity of important enzymes in the body (cytochrome [CYP] P450s) that are involved in drug clearance. This study used a physiologically based pharmacokinetic computer model to investigate the potential impact of increased IL-6 levels on CYP450 enzymes to determine subsequent impact on drugs that are metabolized by CYP450 enzymes. The results showed no predicted interaction between median levels of IL-6 observed in patients and CYP substrates (such as caffeine and omeprazole) with talquetamab. In a simulation that assessed higher (maximum) IL-6 levels observed in patients, the predicted impact of IL-6 was minimal to weak for most of the CYP substrates assessed. The effect on CYP450 enzymatic activity was highest from initiation of talquetamab step-up dosing up to 7-9 days after the first treatment dose of talquetamab. These results suggest that, in this treatment time period, elevated IL-6 levels due to talquetamab-induced cytokine release syndrome have limited impact on drugs that are CYP substrates that may be used in conjunction with talquetamab, but that the concentration and toxicity of these drugs should be monitored and the dose of CYP substrate adjusted as required.

Preclinical activity of allogeneic SLAMF7-specific CAR T-cells (UCARTCS1) in multiple myeloma

BackgroundAutologous BCMA-specific CAR T-cell therapies have substantial activity in multiple myeloma (MM). However, due to logistical limitations and BCMAlow relapses, there is a need for alternatives. UCARTCS1 cells are ‘off-the-shelf’...

Selinexor Enhances Natural Killer Cell Function Against Multiple Myeloma Cells

Introduction: Natural killer (NK) cells are innate immune effector cells which can mediate antibody dependent cellular cytotoxicity (ADCC) and lyse cancer cells. Harnessing NK cell function against multiple myeloma (MM) is of high interest and multiple strategies are currently under development. Exportin-1 (XPO1) is a nuclear export protein which inhibits the function of tumour suppressor proteins and facilitates oncogene mRNA translation. Selinexor is a first-in-class XPO1 inhibitor approved for the treatment of relapsed or refractory MM that induces cancer cell apoptosis. In addition, selinexor has recently been shown to promote NK cell cytotoxicity against B cell lymphoma cells. This study aimed to determine whether selinexor augments NK cell activation against MM cells both alone, and in combination with the anti-CD38 antibody daratumumab. Methods: A panel of MM cell lines (MM.1S, U266 and RPMI-8226) were incubated with clinically relevant concentrations of selinexor for 24 hours, then either assessed for expression of ligands for NK cell receptors (KIR, NKG2A, NKG2D and NKp46), or co-cultured with primary human NK cells ± daratumumab. Following co-culture, NK-mediated lysis of MM cells was assessed by propidium iodide staining and NK cell degranulation assessed by CD107a expression. Results: Selinexor pre-treatment of MM cells significantly increased NK cell degranulation (p<0.05) and enhanced NK specific lysis of MM targets (p<0.05). Furthermore, selinexor pre-treatment significantly (p<0.05) enhanced NK cell activation in combination with daratumumab compared to daratumumab alone. Importantly, selinexor did not alter surface expression of CD38 on MM cell lines. To investigate the mechanism behind this enhanced NK cell function, we assessed the effect of selinexor on the expression of NK cell ligands by MM cells. Selinexor increased the expression of ULBP1/2/5/6 (2-fold, p<0.001) which bind to the NK cell activating receptor NKG2D. In addition, selinexor decreased MM cell expression of HLA-E (30% decrease, p<0.05) which binds to the inhibitory NK receptor NKG2A. Conclusions: These data suggest that selinexor may promote the efficacy of NK targeted therapeutics in multiple myeloma.

Single-Cell Multi-Ome Analysis Reveals Novel Molecular Mechanisms Underlying Subclonal Response to Survivin Inhibition in Relapsed/Refractory Multiple Myeloma

Multiple myeloma is the second-most common hematological malignancy in the US that remains a challenging disease to cure despite recent advancements in treatment strategies. Drug resistance is a major cause of concern in myeloma chemotherapy. We have created an optimization-regularization-based computational prediction method called secDrug that identified several novel secondary drug (‘secDrug‘) combinations against drug-resistant myeloma (relapsed/refractory multiple myeloma or RRMM). Top among our predicted secDrugs includes the survivin inhibitor, YM155. Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has dual roles in mitosis and apoptosis. YM155 inhibits survivin at the transcriptional level by inhibiting the survivin gene promoter. Several studies have demonstrated the efficacy of YM155 in solid tumors and heme malignancies, including myeloma. However, the precise mechanism of action is not yet known. MM cell lines express high levels of IAPs. We observed high in vitro cytotoxicity of YM155 as a single agent (IC 50 range 0.41 to 12.85nM) as well as in combination with the primary drugs like proteasome inhibitors and Immunomodulatory drugs (IMiDs) against >15 human myeloma cell lines representing innate/refractory and acquired/emerging (relapse) resistance. Next, we explored the molecular mechanisms underlying the efficacy of YM155 using pre-vs-post-treatment tumor whole-transcriptome analysis (RNAseq). The top differentially regulated genes include DDIT3 (CHOP), ATF3, TXNDC5 SEC24D, and DHX15 ( Figure 1A). Ingenuity Pathway Analysis (IPA) predicted that, in addition to regulating apoptosis, survivin inhibition in myeloma significantly upregulated unfolded protein response and downregulated vehicle trafficking and genes associated with immune cell function. Further, we performed single-cell transcriptomics (scRNAseq; 10X Genomics) analysis to explore treatment-induced changes to the subclonal architecture. Our scRNAseq results revealed a distinct shift in clusters representing myeloma single-cell subpopulations following YM155 treatment ( Figure 1B). For example, Cluster 1 (53.11%), characterized by high expression of cell survival markers Ki67 and MYC, was pre-dominant in untreated myeloma cells, while Cluster 4 (52.19%) was the primary subclone in post-treatment tumors. Cluster 3 was shared between the untreated and treated groups. Detailed distinctions between each single-cell cluster will be presented. Furthermore, our scRNAseq data alluded to the benefits of combining survivin-inhibitors and Hsp90-inhibitors based on the expression of drug target genes. Hsp90 inhibitors indirectly target survivin by disrupting the interaction between survivin and Hsp90, resulting in a destabilized survivin protein that ultimately results in survivin degradation. In fact, we have earlier shown the effectiveness of Hsp90 inhibitors (17-AAG, AICAR, and CCT018159) as potent secDrugs against RRMM. Finally, high-dimensional mass cytometry or CyTOF analysis using heavy metal-conjugated proteins in patient bone marrow-derived primary myeloma cells ( ex vivo model system) showed that YM155 induced dose-dependent reduction of several protein markers implicated in cell survival and chemotherapeutic resistance in multiple myeloma, including myc, Ki-67, CD28, CD81, and CD274 (PD-L1). Previous studies have shown that CD28 and CD81 are high-risk immunophenotypes associated with significantly worse prognosis in patients with myeloma. PD-L1 (Programmed death-ligand 1) is a cell-surface glycoprotein expressed in a large number of malignancies that is involved in the termination of immune response and immune evasion. An earlier study has shown that YM155 enhances daratumumab-mediated cellular lysis of multiple myeloma cells. Our RNAseq and CyTOF results thus suggest a potential basis of synergy between YM155 with immunotherapies approved for myeloma, which we will test further. Our approach thus integrates in silico prediction with single-cell multi-ome analysis to identify molecular mechanisms potentially underlying subclonal response to novel combination therapy candidates (secDrugs) for the treatment of RRMM.

Preclinical Activity of Allogeneic CS1-Specific CAR T-Cells (UCARTCS1) in Multiple Myeloma

Introduction: The substantial activity of chimeric antigen receptor (CAR) T-cells in multiple myeloma (MM) has recently been shown by the success of BCMA-CAR T-cell therapy. Nevertheless the difficulties in (timely) generation of CART cells for each patient, the limited availability of manufacturing slots, and the frequent BCMAlow MM relapses after therapy urge the investigators to search for i) alternative, "off the shelf", available sources of CAR T-cells and ii) alternative target antigens than BCMA. UCARTCS1 cells are "off the shelf" allogeneic CAR T-cells derived from healthy donors targeting CS1 (SLAMF7), which is a highly attractive target, due to its overexpression on MM cells. Furthermore, the genes coding for the TCRα chain of the TCRab receptor and for CS1 are disrupted in UCARTCS1 cells using Cellectis' TALEN® gene-editing technology. These modifications aim to minimize the potential risk for allo-TCR-mediated graft-versus-host disease (GvHD) upon infusion in patients and diminish CS1-mediated fratricide. In this study, we evaluated the preclinical activity of UCARTCS1 in MM cell lines, in bone marrow (BM) samples obtained from MM patients and in a MM mouse model. We also investigated the potential impact of previous therapy and tumor characteristics on the in vitro efficacy of UCARTCS1. Methods: Luciferase-transduced MM cell lines with different expression levels of CS1 were incubated with UCARTCS1 cells or control (non-transduced CS1/TCRαβ double knock-out) T-cells at different E:T ratios for 24 hours. MM cell lysis was assessed by bioluminescence. Anti-MM activity of UCARTCS1 was also evaluated in 25 BM samples obtained from newly diagnosed (n=10; NDMM), daratumumab-naïve relapsed/refractory patients (n=10; RRMM; median of 3 prior therapies) and from daratumumab-refractory patients (n=5; DRMM; median of 3 prior therapies) in 24-hour flow cytometry-based cytotoxicity assays. The results were correlated to CS1 expression levels of MM cells, Treg frequency, and several other patient characteristics such as treatment history. Results: UCARTCS1 induced strong CS1 CAR-mediated and dose-dependent lysis of different MM cell lines expressing variable levels of CS1 (MM1s, L363, UM9, U266). UCARTCS1 cells also effectively lysed primary MM cells in a dose-dependent and CAR-mediated manner, with a mean maximal lysis of 96.9% (Figure 1). In these BM samples, UCARTCS1 cells also targeted CS1+ non-malignant hematopoietic cells, such as CD4+ T-cells, CD8+ T-cells, B cells, and NK cells, while sparing the CS1low fraction in each cell subset. However, the lysis of normal cells was less efficient than MM cell lysis, which could be explained by substantially lower CS1 expression on normal cells compared to MM cells (Figure 2). CS1 expression on MM cells and UCARTCS1 cytotoxic activity were both comparable in the different patient subgroups. In addition, baseline tumor and immune characteristics, such as the expression level of CS1 on MM cells, the tumor load (MM cell percentage in BM), or frequency of Treg cells in the BM did not affect CAR T-cell mediated cytotoxicity. Finally, UCARTCS1 exhibited durable dose-dependent anti-MM activity in mouse xenograft models after i.v. injection of CAR T-cells compared to mice treated with CAR- control T-cells. Conclusion: UCARTCS1 has potent anti-MM activity against MM cell lines and primary MM cells, as well as in a MM xenograft model. There was no difference in activity between heavily pretreated and newly diagnosed patients, indicating that mechanisms of resistance to prior therapies do not result in reduced susceptibility to UCARTCS1-mediated lysis in vitro. UCARTCS1 was also effective in samples from patients with relatively low CS1 expression, with high tumor burden, or with high Treg numbers. These data support the ongoing phase 1 clinical trial with UCARTCS1 in heavily pretreated MM patients (NCT04142619). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

HDAC Inhibition Involves CD26 Induction on Multiple Myeloma Cells Via the c-Myc/Sp1-Mediated Prompter Activation and Overcomes Therapeutic Resistance By Humanized Antibody

Background: CD26, a 110-kDa transmembrane glycoprotein, which is expressed on several tumor cells including malignant lymphoma has been implicated in tumorigenesis, whereas, little is known about its roles in plasma cell malignancies. Recently, we have identified that CD26 is uniformly and intensely expressed in osteoclasts, while its expression in plasma cells of patients with multiple myeloma (MM) reveals heterogenous due to its cellular diversity or immune escape, leading to a marked heterogeneity of response to humanized anti-CD26 monoclonal antibody (mAb) therapy in MM. Decreased expression levels of CD26 in MM cells are one of the mechanisms underlying innate and acquired resistance to CD26mAb therapy in MM. Aim: Here, we clarify the potential impact of epigenetic modification by HDAC inhibition (HDACi) with isoform selective- or broad- inhibitors on the regulation of CD26 expression in MM cells and elucidate its mechanisms, thereby affecting the performance of CD26mAb in refractory MM. Methods and Results: Immunostaining of bone marrow tissues of MM showed that CD26pos/CD138pos plasma cells were detected in several patients but not in others. First, we investigated the impact of HDACi on CD26 expression of MM cells. Although the cell surface expression of CD26 was relatively low or not detected on 5 MM cell lines (KMS26, KMS27, KMS28, KMS11, RPMI8226), cultured alone, the increased expression in CD26 levels of MM cells was detectable within 24 hr of the treatment with HDAC1i; FK228, HDAC3i; BG45, MS-275, RG2833 or HDAC6i; nexturastat A, tubastatin A, ACY-1215 as well as broad HDACi; LBH-589, SAHA by flow cytometry. Its expression increased further during the exposure to each HDACi and its maximum increase was observed at 72 hr of the treatment. Then, subsequent removal of HDACi for 48 hrs resulted in a decline of CD26 expression on MM cells to reduced or baseline levels. In addition, the levels of CD26mRNA were concomitantly augmented, which was paralleled with an increase in the induction of CD26 protein and its DPPIV enzymatic activity in MM cells. We also examined the effect of each HDACi on the viability of CD26neg MM cells in the absence or presence of CD26mAb.CD26mAb alone did not induce lysis of CD26neg MM cell lines at any doses, whereas, combining each HDACi plus CD26mAb at 10 μg/ml synergistically facilitated lysis of CD26neg MM cells via direct effects as well as NK cell- mediated ADCC by CD26mAb. Of note, HDACi plus CD26mAb in combination readily augmented lysis of CD26neg cell populations, refractory to HDACi or CD26mAb alone. Next, gene expression profiles in KMS11, KMS27 and RPMI8226, left untreated or treated with LBH589 or RG2833 revealed that Myc is one of the overlapped genes, altered in its expression. The 5'-flanking region of human CD26 gene, representing the promoter activity contains potential binding sites of several transcriptional factors including Sp1 and c-Myc. So, further to elucidate the mechanisms by which CD26 expression is induced in MM cells by HDACi, we examined the epigenetic status in histone and non-histone at the CD26 promoter of MM cells with the treatment of HDACi. First, ChIP-qPCR demonstrated that the acetylation in histone 3 lysine 27 is increased at the CD26 promoter of 5 MM cell lines after exposure to each HDACi, suggesting parts of mechanisms in the CD26 promoter activation. Moreover, the expression level of c-Myc was time-dependently decreased in KMS11, KMS27 and RPMI8226 with the treatment of LBH-589 or RG2833 both at mRNA and protein levels and was significantly down-regulated after 12 to 24 hr of exposure. On the other hand, the acetylation of c-Myc on K323 was significantly increased after 12 to 24 hr of exposure to each HDACi. ChIP-qPCR revealed that the recovery % IP/INPUT showing the binding of c-Myc to the CD26 promoter was exposure time-dependently reduced in KMS11, KMS27 and RPMI8226 by the exposure to LBH589 or RG2833. Namely, in the absence of HDACi, c-Myc was present on the CD26 promoter via binding to Sp1 on the proximal G-C box and repressed CD26 promoter in MM cells, whereas, in the presence of HDACi, c-Myc was highly detached from Sp1 on the CD26 promoter after 12 to 24 hr with its increased acetylation, leading to activate CD26 promoter and initiate CD26 transcription in MM cells. Conclusion: HDACi not only shows anti-MM activity itself but sensitizes MM cells with CD26 antigen loss to CD26mAb via c-Myc/Sp1-mediated CD26 induction and augments its cytotoxicity.

γ-secretase inhibitors augment efficacy of BCMA-targeting bispecific antibodies against multiple myeloma cells without impairing T-cell activation and differentiation

We here defined the impacts of γ-secretase inhibitors (GSIs) on T-cell-dependent BCMA-specific multiple myeloma (MM) cell lysis and immunomodulatory effects induced by bispecific antibodies (BisAbs). GSIs-induced membrane BCMA (mBCMA) accumulation reached near maximum within 4 h and sustained over 42h-study period on MM cell lines and patient MM cells. GSIs, i.e., 2 nM LY-411575 or 1 μM DAPT, robustly increased mBCMA densities on CD138+ but not CD3+ patient cells, concomitantly with minimum soluble/shed BCMA (sBCMA) in 1 day-culture supernatants. In ex vivo MM-T-cell co-cultures, GSIs overcame sBCMA-inhibited MM cell lysis and further enhanced autologous patient MM cell lysis induced by BCMAxCD3 BisAbs, accompanied by significantly enhanced cytolytic markers (CD107a, IFNγ, IL2, and TNFα) in patient T cells. In longer 7 day-co-cultures, LY-411575 minimally affected BCMAxCD3 BisAb (PL33)-induced transient expression of checkpoint (PD1, TIGIT, TIM3, LAG3) and co-stimulatory (41BB, CD28) proteins, as well as time-dependent increases in % effector memory/central memory subsets and CD8/CD4 ratios in patient T cells. Importantly, LY41157 rapidly cleared sBCMA from circulation of MM-bearing NSG mice reconstituted with human T cells and significantly enhanced anti-MM efficacy of PL33 with prolonged host survival. Taken together, these results further support ongoing combination BCMA-targeting immunotherapies with GSI clinical studies to improve patient outcome.

Abstract 5473: Isoform-selective HDAC inhibition up-regulates CD26 expression on multiple myeloma cells and augments cytotoxic efficacy by humanized monoclonal antibody

Abstract Objective: CD26, a 110-kDa transmembrane glycoprotein, which is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis, whereas, its roles in plasma cell malignancies remain elusive. Recently, we have identified that CD26 is uniformly and intensely expressed in osteoclasts, while its expression in plasma cells of patients with multiple myeloma (MM) reveals heterogenous. Decreased expression levels of CD26 in MM cells are one of the mechanisms underlying resistance to humanized anti-CD26 monoclonal antibody (mAb) therapy. In the present study, we clarify the impact of epigenetic modification by HDAC inhibition (HDACi) with isoform-selective or broad inhibitors on the regulation of CD26 in MM cells and its mechanisms, thereby affecting the performance of humanized CD26mAb. Methods and Results: Immunostaining of bone marrow tissues of MM showed that CD26pos/CD138pos plasma cells were detected in several patients but not in others. So, first, we investigated the impact of HDACi on CD26 expression of MM cells. Although the cell surface expression of CD26 was relatively low or not detected on 5 MM cell lines (KMS26, KMS27, KMS28, KMS11, RPMI8226), the increased expression in CD26 levels was detectable within 24 h of the treatment with HDAC1i; FK228, HDAC3i; BG45, MS-275, RG2833 or HDAC6i; nexturastat A, tubastatin A, ACY-1215 as well as broad HDACi; LBH-589, SAHA. It increased further and maximum increase was observed at 72 h of the treatment by each HDACi. Then, subsequent removal of HDACi resulted in a decline of CD26 expression on MM cells to pre-treated levels. In addition, the levels of CD26mRNA were concomitantly enhanced, which was paralleled with an increase in the induction of CD26 protein in MM cells. Next, we examined the effect of HDACi on the viability of CD26neg MM cells in the absence or presence of CD26mAb. The mAb alone did not induce lysis of CD26neg MM cell lines at any doses, whereas, combining HDACi plus CD26mAb (10 μg/ml) synergistically facilitated lysis of CD26neg MM cells via direct effects as well as NK cell-mediated ADCC by mAb. Of note, HDACi plus CD26mAb in combination readily augmented lysis of CD26neg cell populations, refractory to HDACi or mAb alone. Furthermore, to elucidate the mechanisms of CD26 up-regulation in MM cells by HDACi, we performed chromatin immunoprecipitation assay on CD26 gene promoter. Each HDACi increased acetylation of histone 3 lysine 27 (H3K27Ac), concomitant with enhanced binding of Sp1 at the 5’ flanking region of the CD26 gene containing Sp1 binding sites in CD26neg MM cells, which is suggestive of transactivation of CD26 gene by HDACi targeting HDAC1, 3 or 6. Conclusion: Combination with isoform-selective HDACi not only shows anti-MM activity but supports as immunopotentiators by sensitizing CD26neg MM cells to CD26mAb and augment its cytotoxicity against MM cells. Citation Format: Hiroko Nishida, Mari Fujiwara, Mutsumi Hayashi, Michiie Sakamoto. Isoform-selective HDAC inhibition up-regulates CD26 expression on multiple myeloma cells and augments cytotoxic efficacy by humanized monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5473.

Health-related quality of life in patients with relapsed/refractory multiple myeloma (RRMM) treated with teclistamab, a B-cell maturation antigen (BCMA) x CD3 bispecific antibody: Patient-reported outcomes in MajesTEC-1.

8033 Background: As multiple myeloma (MM) negatively affects patients’ (pts) health-related quality of life (HRQoL), assessment of patient-reported outcomes (PROs) in addition to clinical outcomes is important. Teclistamab (tec; JNJ-64007957) is an off-the-shelf bispecific antibody that redirects CD3+ T cells to mediate T-cell activation and subsequent lysis of BCMA-expressing MM cells. Initial results from the pivotal cohort of the phase 1/2 MajesTEC-1 study demonstrated that tec was well tolerated with encouraging efficacy in pts who received ≥3 prior lines of treatment (LOT; including a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody). Here we report PROs from this cohort. Methods: Pts (aged ≥18 years) had documented RRMM (International Myeloma Working Group criteria), progressive/measurable disease, and had previously received ≥3 prior LOT; prior anti-BCMA treatment (tx) was not allowed. Pts received weekly subcutaneous tec at the recommended phase 2 dose (1.5 mg/kg with step-up doses of 0.06 and 0.3 mg/kg). PROs were assessed at screening and every even cycle (cycles 2–8 reported here) using the EORTC QLQ-C30 (range: 0–100; higher scores indicate better global health status [GHS] but greater symptom severity [symptom scales]) and the EuroQol 5-dimensional descriptive system (visual analog scale [VAS] range: 0 [worst imaginable health state] to 100 [best imaginable]). Tx effect was assessed by a mixed-effects model with repeated measures; the proportion of pts with meaningful improvement was defined as a change ≥10 points. Time to worsening was determined using the Kaplan-Meier estimate. Results: A total of 110 pts were included (median follow-up: 7.8 mos). Overall PRO compliance rates were high (baseline [BL]: 85–90%; cycles 2–8: 80–94%). Tec improved overall HRQoL as evidenced by improvements in GHS scores (cycles 2–8) and reduction in pain (-4.2 [cycle 2] to -15.1 [cycle 8]; Table), with no overall change in physical functioning and fatigue. The proportions of pts with meaningful improvements from BL at cycle 8 were GHS: 50%; physical functioning: 35%; pain: 65%; fatigue: 73%; 50% of pts reported meaningful improvement in their overall health (VAS). Median time to improvement from baseline was ̃1.5 months (with nausea/vomiting and fatigue taking longer to improve), while median time to worsening (all symptoms) ranged from 2 months to not estimable. Conclusions: Consistent with clinical outcomes, pts treated with tec reported rapid, clinically meaningful improvements in HRQoL. Clinical trial information: NCT04557098. [Table: see text]

IBI379, a novel B cell maturation antigen/CD3 bispecific T-cell engager, displays high antitumor efficacy in preclinical models of multiple myeloma

Multiple myeloma (MM) is a hematological malignancy that results from the malignant proliferation of plasma cells in the bone marrow. B cell maturation antigen (BCMA) is highly selectively expressed in malignant plasma cells and is a novel therapeutic target for MM. Here, we developed a bispecific T cell engager, IBI379, that targets BCMA and CD3, and investigated its antitumor efficacy against MM. IBI379 showed strong binding affinity with both BCMA and CD3, which triggered T cell activation, proliferation, and cytokine release. An in vitro study demonstrated that IBI379 induced the lysis of MM cells expressing differing levels of BCMA on the cell surface. Administration of IBI379 in H929 or Daudi-BCMA cell xenograft mouse models significantly inhibited tumor growth without inducing body weight loss. The mechanism of action study revealed the accumulation of CD4+CD8+ T cells and granzyme B-positive T cells in tumors that were treated with IBI379. Moreover, administration of low dose of IBI379 in cynomolgus monkeys was well-tolerated and induced the depletion of BCMA+ B cells and a mild transient increase of cytokines. Collectively, these results demonstrate that IBI379 is a highly potent therapeutic strategy for depleting BCMA-positive B cells and is a promising approach for the treatment of MM.

A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

Phase 1b Results for Subcutaneous Talquetamab Plus Daratumumab in Patients with Relapsed/Refractory Multiple Myeloma

Phase 1b Results for Subcutaneous Talquetamab Plus Daratumumab in Patients with Relapsed/Refractory Multiple Myeloma

CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu

CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu

Preclinical Assessment of CDR101 - a BCMAxCD3xPD-L1 Trispecific Antibody with Superior Anti-Tumor Efficacy

BCMAxCD3 targeting therapies have demonstrated anti-myeloma activity, and high minimal residual disease negativity rates can be achieved with this approach in heavily pre-treated patients with relapsed or refractory multiple myeloma (RRMM). Despite these promising clinical results, patients eventually develop resistant disease and relapse. Thus, there is a high need for novel BCMA therapies that can evade the resistance mechanisms and provide more durable responses. Recently, we reported on the promising activity of the Local Activator and T cell Engager (LocATE) technology, a trispecific molecule that targets CD3, BCMA and PD-L1, redirecting T cells to multiple myeloma (MM) cells while selectively counteracting PD-L1/PD-1 induced immunosuppression at the immune synapse (ASH, 2020). Here we present CDR101, an optimized LocATE candidate with potential for clinical development.First, we analyzed the ability of CDR101 to induce PBMC-mediated cytotoxicity in two MM cell-lines expressing BCMA (U-266 and NCI-H929) and compared it to four BCMAxCD3 bispecific formats currently in clinical development (a half-life extended BCMAxCD3 BiTE, a BCMA-TCB, and two different BCMAxCD3 bispecific monoclonal antibodies) alone or in combination with a separate PD-L1 blocking antibody. CDR101 resulted in at least 10-fold increased T cell-mediated target cell lysis compared to control BCMAxCD3 bispecifics. Strikingly, CDR101 also resulted in increased MM cell killing when compared to free, independent combinations of BCMAxCD3 bispecifics and the PD-L1 inhibitor. These results, together with the observation that MM cells upregulate the expression of PD-L1 in response to treatment with BCMAxCD3 bispecifics, suggest that the superior effect of CDR101 could be attributed to preferential and highly selective inhibition of the PD-1/PD-L1 axis at the cellular interaction within the immune synapse.Next, bone marrow aspirates from newly diagnosed and RRMM patients were treated with increasing concentrations of CDR101 or a BCMAxCD3 bispecific control. After 24h of incubation, percentage of viable CD138-positive cells and activation status of autologous T cells were analyzed by FACS. Overall, CDR101 potently induced lysis of primary MM cells independently of the E:T ratio (range of E:T ratio between 1.3:1 and 33:1). CDR101 achieved higher target cell killing in all samples compared to the bispecific control, with at least 2-fold difference in 3 out of 4 samples at the highest concentration tested. Concomitantly, CDR101 induced a dose-dependent increase of the T cell activation marker CD25, corroborating the ability of CDR101 to counteract PD-L1/PD-1 induced immunosuppression.In vivo anti-tumor activity of CDR101 was evaluated using a human MM (NCI-H929) xenograft model in NPG mice. Treatment with four different doses of CDR101 or BCMAxCD3 bispecific control demonstrated that CDR101 induced stronger and more durable responses compared to the bispecific control leading to complete tumor regression in 55 out of 60 mice at the last day of treatment (day 29) with no relapse until the end of the observation time (day 41).Collectively, CDR101 demonstrated that targeting BCMA with simultaneous blockade of PD-L1 leads to improved myeloma cell killing compared to clinically validated therapies. In contrast to high-affinity PD-L1 immune checkpoint inhibitors, CDR101 selectively inhibits PD-L1 at the immune synapse preventing on-target off-tumor effects. This is expected to translate into a decreased incidence of immune related adverse events (irAEs) and better efficacy arguing for a high clinical potential and swift translation into the clinic. DisclosuresVrohlings: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Jungmichel: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Senn: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Howald: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Schleier: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Scheifele: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Wendelspiess: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Richle: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Merten: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Lenherr-Frey: CDR-Life Inc: Current Employment, Current holder of stock options in a privately-held company. Leisner: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA. Borras: CDR-Life Inc: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

OAB-033: Loss-of-function of GABARAP drives tumor resistance to bortezomib-induced immunogenic cell death in multiple myeloma

<h3>Background</h3> Resistance to immune approaches poses a major challenge to effective immunotherapy and long-term clinical outcome in multiple myeloma (MM). Here we identified loss-of-function of gamma-aminobutyric acid receptor-associated protein (GABARAP) as a tumor-intrinsic mechanism of resistance to bortezomib (BTZ)-induced immunogenic cell death (ICD), the immunogenic consequence of apoptosis resulting in specific anti-MM immunity via T-cell priming by dendritic cells (DCs). <h3>Methods</h3> We found that BTZ induces ICD in human and murine MM cell lines that is dependent on the exposure of calreticulin (CALR), which drives DCs-mediated phagocytosis of MM cells. DCs-phagocytosis is followed by a specific T cell activation, with a significant increase of CD4+ effector memory (EM), total CD8+, CD8+ EM, and CD8+ terminally differentiated EM cells. Isolated T cells after co-cultures showed the presence of MM specific CTLs that were able to efficiently induce lysis of MM cells. Our results were validated using primary cells from MM patients. Moreover, induction of a protective immune response was confirmed in vivo. Specifically, treatment of 5TGM1 tumors with BTZ induced a tumor regression in a syngeneic model; and injection of live 5TGM1WT two weeks after regression did not result in tumor development, consistent with induction of immunological memory as confirmed by ELISPOT of mouse splenocytes. We identified a specific ICD signature induced by BTZ in mice; and we found that increased expression of the human orthologs of this signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Notably, these functional immunologic sequelae were abrogated after BTZ treatment of CALRKO MM cells both in vitro and in vivo, confirming the obligate role of CALR exposure in the ICD process. <h3>Results</h3> By interrogating the IFM/DFCI dataset and focusing on genes involved in ICD processes that were correlated with MM patients clinical outcome, we found that low levels of GABARAP (chr17p13.1), an autophagy regulator and putative CALR binding partner, negatively impact MM patient clinical outcome (EFS, p=0.0032); even excluding HR patients with 17p deletion (EFS, p=0.018). Interestingly, KMS11 cells carrying monoallelic deletion of GABARAP were resistant to induction of ICD by BTZ; and sensitivity was restored after overexpression of the gene. Moreover, GABARAPKO in 3 ICD-sensitive cell lines abrogated the induction of ICD by BTZ; and add-back experiments by pre-treatment with recombinant CALR or GABARAP overexpression in KO clones restored ICD. Finally, CyTOF confirmed that treatment of GABARAPKO cells with BTZ failed to activate an efficient T cell response. <h3>Conclusions</h3> our study demonstrates that loss-of-function of GABARAP, particularly in HR patients with 17p deletion, contributes to tumor immune evasion and ICD resistance. These studies provide the framework for novel combination treatments to restore anti-MM immunity and improve patient outcome in HR MM.

A First in Class APRIL Fully Blocking Antibody Targets Novel Immune Regulation of APRIL in Multiple Myeloma: Further Therapeutic Implication

A proliferation inducing ligand (APRIL), a critical paracine factor for human multiple myeloma (MM), is mainly secreted by myeloid cells, including macrophages and osteoclasts in the bone marrow (BM) microenvironment. Recently, an antagonistic APRIL monoclonal antibody (mAb) 01A significantly reduced myeloma burden in SCID-hu mice (Blood. 2016;127:3225-3236). This data suggests that the humanized 01A named BION-1301, which also potently (IC50 <1 nM) blocks APRIL binding to its receptors B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator (TACI) is likely to exert anti-MM activity in vivo. Here, we further characterize the novel inhibition by 01A and a hIgG4 chimeric version C4 on multiple immune cells affected by APRIL in the immunosuppressive MM BM milieu. First, using gene-modified RPMI8226 MM cells, 01A potently and selectively blocks growth and viability of APRIL-overexpressing but not BCMA-overexpressing RPMI8226 transfectants. Significantly, 01A inhibits MM cell growth and survival induced by osteoclasts, as a physiologically relevant endogenous source of APRIL. Next, we demonstrate that pretreatment of APRIL decreases MM cell lysis induced by Daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). In addition, APRIL prevents anti-BCMA (J6M0)-induced ADCC against BCMA-expressing MM cell lines in a dose-dependent manner. Conversely, 01A or C4 (a surrogate of BION-1301) reverts APRIL-reduced MM cell lysis induced by J6M0 in the presence of PBMC or NK effector cells. These data indicate that targeting CD38 and BCMA may not be sufficient in MM. To further define the role of APRIL mediating anti-MM immune responses, we show that T cells do not express BCMA while natural T regulatory (Treg) cells significantly express higher TACI when compared with corresponding conventional T cells (Tcon) from the same individual (Abstract #102467). In parallel, detailed gene expression profiling in multiple solid tumors show that the tumor-resident Treg markers are significantly correlated with TACI, along with a strong association of APRIL expression levels with T cell inflamed signature. In addition to natural Treg, MM cell-induced Tregs (iTregs) also showed significantly increased TACI levels when compared with paired conventional T cells (Tcon) from the same individual in ex vivo cocultures. Importantly, APRIL further enhances MM cell-induced iTregs with IL-10-producing and CD15s+[JD2] FOXP3high fractions; conversely, 01A blocks APRIL-induced upregulation of iTreg. Osteoclasts also upregulated iTreg generation induced by MM cells. On the contrary, 01A or C4 partially blocks such induction. Importantly, APRIL directly promotes proliferation and viability of Treg via the induction of CCND1/2, BCL2, and BCL2L1/BCL-xL, which is blocked by 01A or C4. Finally, APRIL enhances the suppressive function of Treg on the proliferation of autologous conventional T cells, which could also be overcome by 01A or C4. Collectively, our data therefore demonstrate that APRIL, via TACI but not BCMA, critically modulates regulatory T cell function; and, importantly, 01A or C4 targets these immune regulatory subsets that have been linked to MM progression via APRIL-dependent manner. Moreover, since patients with active diseases have severe bone lesions induced by hyperactive osteoclasts, inhibiting the APRIL-TACI interaction may also overcome osteoclast-inhibited T cell killing on MM cells. Anti-APRIL Ab (BION-1301) will be entering clinical trial in late 2017 to target the immunosuppressive BM microenvironment and improve patient outcome in MM. DisclosuresDulos:Aduro Biotech Europe: Employment, Equity Ownership. Van Elsas:Aduro Biotech Europe: Employment, Equity Ownership. Richardson:Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees. Anderson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Humanized Anti-CD26 Monoclonal Antibody Has Anti-Myeloma Efficacy for the Treatment of High-Risk Multiple Myeloma

Background: Despite remarkable progress in treatment options of multiple myeloma (MM) over the last decade with proteasome inhibitors and immunomodulatory drugs (IMiDs), most of patients with high-risk multiple myeloma (MM) relapse and ultimately become refractory. Recently, novel monoclonal antibody directly targeting CS1(elotuzumab) or CD38 (daratuzumab) induced durable responses in patients with multidrug-refractory MM. CD26, a 110-kDa transmembrane glycoprotein which is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis, whereas its role in plasma cell malignancies has not been characterized yet. Our recent work has revealed that CD26 is intensely expressed on activated osteoclasts (OCs) in MM. In addition, in vitro studies showed that although CD26 expression is only slightly detected on MM cell lines cultured alone, it is intensely and uniformly expressed on MM cell lines co-cultured with OCs. In this study, we clarify the direct on-tumor and immune mechanism of action with huCD26mAb, a humanized monoclonal IgG1 antibody specifically targeting CD26, alone or in combination with approved agents in high-risk MM.Methods and Results: Immunostaining on bone marrow biopsy specimens showed that CD26 is expressed on plasma cells in several MM patients, but not in those of healthy individuals. So, we first analyzed the effect of huCD26mAb on survival of MM cell lines (KMS18, KMS26, KMS27, KMS28, KMS34, U266). HuCD26mAb had no direct effect on proliferation in any of tested CD26- MM cell lines cultured alone, but it inhibited growth of CD26+ MM cell lines co-cultured with OCs at higher concentrations (>10μg/ml), as measured by the MTT assay. Next, we examined the ability of huCD26mAb to lyse CD26 positive MM cell lines by antibody-dependent cellular cytotoxicity (ADCC) using the calcein-AM release assay. HuCD26mAb, but not isotype control IgG1, triggered ADCC against CD26+ KMS18, KMS26, KMS28, KMS34 and U266 with t(4;14) by natural killer (NK) effector cells in a dose-dependent manner with lytic activity starting at 0.0001μg/ml and maximum lysis at 10μg/ml and in an effector to target (E/T) ratio-dependent manner. Moreover, huCD26mAb dose-dependently induced lysis of lenalidomide-resistant MM.1R via ADCC. We further asked whether pretreatment with conventional or novel agents is able to induce superior ADCC activity by huCD26mAb. CD26+ KMS18, KMS26 and KMS28 were pretreated with bortezomib (3nM), lenamlidomide (0.5μM) or dexamethasone (25nM) overnight. Indeed, subsequent MM cell lysis triggered by huCD26mAb (10μg/ml) was significantly augumented. Treatment with lenalidomide (0.5μM) followed by huCD26mAb (10μg/ml) also markedly reduced the viable MM.1R in the presence of NK effector cells. The synergistic ADCC efficacy of huCD26mAb in combination with lenalidomide resulted in 2.0-fold increase in MM.1R lysis. In addition, pretreatment of NK effector cells with lenalidomide also potentiated ADCC against MM.1R mediated by huCD26mAb in a dose-dependent fashion. Complement dependent cytotoxicity (CDC) of CD26+ MM cells by huCD26mAb was also tested using human serum as a source of complement. However, in the presence of human serum, the incubation with huCD26mAb or isotype IgG1 exerted no effect on MM cell lysis. These results indicate that huCD26mAb (≦10μg/ml) kills CD26+ MM cells by inducing ADCC but not CDC, regardless of sensitivity or resistance to conventional or novel agents. Finally, to further determine whether huCD26mAb alone or combination with lenalidomide affects side population (SP) fractions of drug-resistant MM cells, we first examined CD26 expression in SP and main population (MP) fractions by flow cytometry. Both CD26+ RPMI8226 and KMS11 exhibited SP fractions which equally expressed CD26 at high levels as MP fractions. So, we examined ADCC activity by huCD26mAb (10μg/ml) against these SP fractions, mixed with NK effector cells. Interestingly, although lenalidomide alone did not decrease the ratio of SP fractions, huCD26mAb substantially reduced their ratio and its further reduction was observed with both in combination against CD26+ RPMI8226 and KMS11.Conclusions: HuCD26mAb induced significant anti-MM efficacy chiefly through ADCC. CD26 may be a novel valuable target for antibody therapy and huCD26mAb, alone or in combination, may be a promising immunotherapeutic strategy for the treatment of high-risk MM. DisclosuresMorimoto:Y's Therapeutics: Consultancy, Research Funding. Yamada:Y's therapeutics: Consultancy, Research Funding.

Potent Ex Vivo Anti-Tumor Activity in Relapsed Refractory Multiple Myeloma Using Novel DR5-Specific Antibodies with Enhanced Capacity to Form Hexamers upon Target Binding

Hyperclustering of Death Receptor 5 (DR5) after binding of its ligand TRAIL (TNF-related apoptosis-inducing ligand), induces apoptosis. As cancer cells are more sensitive for TRAIL-induced apoptosis than nonmalignant cells, targeting DR5 with agonistic monoclonal antibodies (mAbs) has been evaluated as a potentially effective therapy for several cancer types. Unfortunately, clinical results with conventional DR5-targeting mAbs have been disappointing due to lack of efficacy.To improve antibody-mediated clustering of DR5 on cancer cells we applied the novel HexaBody® technology, which is based on the natural concept that, upon binding to antigens on a cell surface, immunoglobulins (IgGs) can organize into ordered hexamers through Fc-Fc interactions. By introducing a single point mutation at specific residues in the IgG1 Fc domain, hexamer formation could be significantly enhanced. Accordingly, Hx-DR5-01/05 (GEN1029) was developed. Hx-DR5-01/05 is a mixture of two non-competing DR5-specific antibodies, with enhanced capacity to form hexamers upon target binding due to an E430G mutation in their Fc domains. Thus, Hx-DR5-01/05 induces potent DR5 agonist activity through a combination of hexamer formation and dual-epitope targeting. In contrast to conventional DR5 specific antibodies, Hx-DR5-01/05-mediated tumor cell kill is independent of Fc gamma receptor-mediated crosslinking.Here, we explored the potential utility of Hx-DR5-01/05 in multiple myeloma (MM) by analyzing cytotoxic activity against MM cell lines and a large panel of primary MM cells derived from patients with newly diagnosed or relapsed refractory disease. Hx-DR5-01/05 induced effective cytotoxicity in MM cell lines, whereas the same antibodies without the E430G mutation did not, underscoring the important contribution of hexamer formation for DR5 agonist activity of Hx-DR5-01/05.Similarly, ex vivo assays using primary MM cells present in the bone marrow mononuclear cells of MM patients revealed superior lytic activity of Hx-DR5-01/05 against primary MM cells as compared to its wild-type counterpart. Overall, the lysis levels of primary MM cells displayed a weak but significant correlation with their DR5 expression levels. Importantly, MM cell lysis levels equal or above 20% were observed in a significantly higher fraction of relapsed refractory patients (74%) compared to those who are newly diagnosed (30%), which correlated with significantly higher DR5-expression levels of MM cells from relapsed/refractory compared to newly-diagnosed patients. In conclusion, our ex vivo results indicate superior therapeutic potential of Hx-DR5-01/05 as compared to conventional DR5 antibodies, with the highest activity in relapsed/refractory MM patients. DisclosuresOverdijk:Genmab: Employment. Breij:Genmab: Employment. Chamuleau:Genmab: Research Funding; Gilead: Research Funding. Lokhorst:Genmab: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; OncoImmune: Research Funding. Mutis:Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; OncoImmune: Research Funding; Novartis: Research Funding; Celgene: Research Funding; Gilead: Research Funding.

Preclinical activity and determinants of response of the GPRC5DxCD3 bispecific antibody talquetamab in multiple myeloma

Cell surface expression levels of GPRC5D, an orphan G protein–coupled receptor, are significantly higher on multiple myeloma (MM) cells, compared with normal plasma cells or other immune cells, which renders it a promising target for immunotherapeutic strategies. The novel GPRC5D-targeting T-cell redirecting bispecific antibody, talquetamab, effectively kills GPRC5D+ MM cell lines in the presence of T cells from both healthy donors or heavily pretreated MM patients. In addition, talquetamab has potent anti-MM activity in bone marrow (BM) samples from 45 patients, including those with high-risk cytogenetic aberrations. There was no difference in talquetamab-mediated killing of MM cells from newly diagnosed, daratumumab-naïve relapsed/refractory (median of 3 prior therapies), and daratumumab-refractory (median of 6 prior therapies) MM patients. Tumor cell lysis was accompanied by T-cell activation and degranulation, as well as production of pro-inflammatory cytokines. High levels of GPRC5D and high effector:target ratio were associated with improved talquetamab-mediated lysis of MM cells, whereas an increased proportion of T cells expressing PD-1 or HLA-DR, and elevated regulatory T-cell (Treg) counts were associated with suboptimal killing. In cell line experiments, addition of Tregs to effector cells decreased MM cell lysis. Direct contact with bone marrow stromal cells also impaired the efficacy of talquetamab. Combination therapy with daratumumab or pomalidomide enhanced talquetamab-mediated lysis of primary MM cells in an additive fashion. In conclusion, we show that the GPRC5D-targeting T-cell redirecting bispecific antibody talquetamab is a promising novel antimyeloma agent. These results provide the preclinical rationale for ongoing studies with talquetamab in relapsed/refractory MM.