Despite several approved therapies, multiple myeloma (MM) remains an incurable disease with high unmet medical need. " Off-the-shelf" T-cell bispecific antibodies (TCBs) targeting B-cell maturation antigen (BCMA) and G protein-coupled receptor class C group 5 member D (GPRC5D) have demonstrated high objective response rates in heavily pretreated patients with MM; however, primary resistance, short duration of response, and relapse driven by antigen shift frequently occur. Although GPRC5D represents the most selective target in MM, recent fi ndings indicate antigen loss occurs more frequently than with BCMA. Thus, anti-GPRC5D immunotherapies must hit hard during a short period of time. Here, we characterize forimtamig, a novel GPRC5D-targeting TCB with 2+1 format. Bivalent binding of forimtamig to GPRC5D confers higher affinity than classical 1+1 TCB formats correlating with formation of more stable immunological synapses and higher potency in tumor cell killing and T-cell activation. Using an orthotopic mouse model of MM, forimtamig recruited T effector cells to the bone marrow and induced rapid tumor killing even after the introduction of step-up dosing to mitigate cytokine release. Combination of forimtamig with standard-of-care agents including anti-CD38 antibodies, immunomodulatory drugs, and proteasome inhibitors improved depth and duration of response. The combination of forimtamig with novel therapeutic agents including BCMA TCB and cereblon E3 ligase modulatory drugs was potent and prevented occurrence of GPRC5D-negative tumor relapse. Forimtamig is currently being evaluated in phase 1 clinical trials in patients with relapsed and refractory MM for monotherapy and in combination treatments. This trial was registered at www.ClinicalTrials.gov as #NCT04557150.

ABSTRACT:Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.

IntroductionThe serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy.MethodsTo assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both in vitro and in vivo.ResultsIn a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. In vivo, STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti–PD-L1 antibody in the MCA205 model.ConclusionsIn summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses in vitro and in vivo. This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy.

The development of Venetoclax (Ven) and Azacytidine (Aza) as treatment option in Acute Myeloid Leukemia (AML) has been a breakthrough especially for patients unfit for intensive chemotherapy (IC).Ven/Aza induced complete remission (CR) in 36.7% of newly diagnosed elderly AML patients [1].However, long term response rates remain poor with a median event-free survival of 9.8 months and a median overall survival of 14.1 months [1], due to persistence of measurable residual disease (MRD) that drives relapse.Novel therapeutic strategies are therefore urgently needed.Data from B-cell malignancies have demonstrated that T-cell based immunotherapy platforms, e.g.T-cell bispecific antibodies (TCBs) or chimeric antigen receptor (CAR) engineered T cells, are able to induce long-term remission and even cure in this patient cohort.Treatment with Blinatumomab in B-cell precursor acute lymphoblastic leukemia resulted in significant elimination of MRD in low-burden disease [2].Therefore, utility of immunotherapies such as TCBs post Ven/Aza could be envisaged to eradicate the MRD pool in AML and to improve survival rates in this patient population.This concept is further supported by preclinical data that demonstrated a Venetoclax induced increase in reactive oxygen-species (ROS) production in T cells, which resulted in enhanced T-cell effector function [3].In addition, Azacytidine increased the susceptibility of AML cells for T-cell mediated lysis [3].Multiple early phase clinical trials with TCBs targeting CD123 (Flotetuzumab and Vibecotamab) or the intracellular tumor antigen Wilms tumor 1 (WT1) presented on HLA-A*02 are currently underway in AML [4-6].In the current report, we investigate the immune modulating impact of Ven/Aza on the efficacy of a WT1-targeted TCB in AML in vitro and in vivo.To this end, we analyzed the PBMC composition of three healthy donors (HD) in vitro by flow cytometry after three days of treatment with 25 nM Ven and 1 µM Aza, reflecting patient serum concentrations [7, 8], and compared to an untreated control.High

T cell bispecific antibodies (TCBs) are a promising new class of therapeutics for relapsed/refractory multiple myeloma. A frequently observed, yet incompletely understood effect of this treatment is the transient reduction of circulating T cell counts, also known as T cell margination (TCM). After administration of the GPRC5D-targeting TCB forimtamig (RG6234), TCM occurred in patients and correlated with cytokine release and soluble B cell maturation antigen decrease. We demonstrate that TCM is accurately represented in the humanized NSG mouse model and occurs at a lower threshold of target expression than systemic cytokine release. Application of whole-mouse tissue clearing and 3D imaging revealed that T cells accumulate in the bone marrow after treatment. We hypothesize that low amounts of targets are sufficient to rapidly redirect T cells upon TCB engagement. Therefore, we propose TCM as a beneficial, highly sensitive and early effect of forimtamig that leads T cells to likely sites of bone marrow tumor lesions.

Introduction: Robust biomarkers of response and resistance to chimeric antigen receptor (CAR) T cell therapy in patients with relapsed/refractory multiple myeloma (RRMM) are lacking.We conducted a longitudinal single-cell multi-omics study to identify factors predicting response to BCMA-directed CAR T cells.Methods: Peripheral blood samples from 10 patients (8 Ide-cel; 2 Cilta-cel) were collected on the day of leukapheresis and 30 days after CAR T cell treatment.Bone marrow biopsies were performed on day 30 after CAR T cell therapy.We used 57 oligonucleotidecoupled antibodies for surface proteome analysis.Libraries for single cell BCR, TCR and RNA were generated using the 10x genomics 5' chemistry.Cell types were annotated with Seurat and WNN.scCODA identified cell type composition changes.InferCNV detected CNVs in malignant plasma cells.Ligand-receptor signaling was inferred with iTalk and CellPhoneDB.CAR T cell in vitro functionality was tested through cytotoxicity assays.For analyses patients were divided by their response to CAR T cell therapy on day 30 after infusion (CR: n=5, no CR: n=5).Results: We sequenced 178,142 cells (median 7,990 cells/sample, range 1,569-10,972 cells) and already observed differences between patients in CR and no CR at leukapheresis.CR patients harbored more CD8+ TEM and NK cells but fewer monocytes.Non-responders showed recurrently higher PIM kinase expression in monocytes, dendritic and NK cells as well as higher protein expression of immune checkpoints on monocytes (CD39) and NK cells (CD94).Cellcell interaction analysis identified inhibitory communication of monocytes with NK and CD8+ T cells in non-responders.Since we detected an immunosuppressive environment in non-responders at leukapheresis, we aimed at characterizing the functionality of manufactured CAR T cells.CAR T cells isolated from patients in CR and no CR at day 7 post-infusion, effectively eliminated MM cells (U-266), indicating that also CAR T cells from non-responders remained functional in vitro.Comparing single-cell transcriptomes of CAR T cells isolated from patients in CR who received Cilta-cel or Ide-cel, we noticed an upregulation of genes associated with cell cycle regulation, exhaustion/senescence, and chemotaxis in Cilta-cel CAR T cells.Surface proteomics revealed that hyperexpanded CAR T cells displayed a more exhausted and senescent phenotype, characterized by higher expression levels of immune checkpoints and NK cell receptors (PD1, CD57, CD94), as well as lower expression levels of

Supplementary Data from Activity and Resistance of a Brain-Permeable Paradox Breaker BRAF Inhibitor in Melanoma Brain Metastasis

Background:Acute Myeloid leukemia is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype.Methods:We characterized bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry and RNA sequencing using computational analysis. In addition, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab (also referred to as RG6292 and RO7296682) or isotype control antibody on regulatory T cells and CD25+ AML cells.Results:Bone marrow composition, in particular the abundance of regulatory T cells and CD25 expressing AML cells, correlated strongly with that of the blood in patients with time-matched samples. In addition, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing a FLT3-ITD mutation or treated with a hypomethylating agent in combination with venetoclax. We adopted a patient-centric approach to study AML clusters with CD25 expression and found it most highly expressed on immature phenotypes. Ex vivo treatment of primary AML patient samples with CD25 Mab, a human CD25 specific glycoengineered IgG1 antibody led to the specific killing of two different cell types, CD25+ AML cells and regulatory T cells, by allogeneic Natural Killer cells.Conclusion:The in-depth characterization of patient samples by proteomic and genomic analyses supported the identification of a patient population that may benefit most by harnessing CD25 Mab's dual mode of action. In this pre-selected patient population, CD25 Mab could lead to the specific depletion of regulatory T cells, in addition to leukemic stem cells and progenitor-like AML cells that are responsible for disease progression or relapse.

Despite novel therapeutic options leading to a substantial increase in survival rates, multiple myeloma (MM) still represents an incurable disease. T cell bispecific antibodies (TCBs) have become a novel therapeutic option for relapsed refractory myeloma (RRMM) patients based on their promising objective response rates (ORR), favorable safety profile and off-the-shelf availability as compared to CAR-T cell therapies. Although BCMA- and GPRC5D-targeted TCBs have been reported to induce deep clinical responses, antigen drift represents a tumor intrinsic resistance mechanism limiting durability of responses 1. Here, we report that the combination of forimtamig, a 2:1 GPRC5D-targeted TCB, with CELMoDs, but not IMIDs, prevents tumor relapse driven by target-negative myeloma cells in a preclinical model of multiple myeloma. In order to mimic acquired TCB resistance in vivo, stem-cell humanized mice were engrafted with NCI-H929 tumors and treated once weekly (QW) with fixed duration forimtamig at 0.1 mg/kg for 6 cycles with a treatment-free follow up of more than 2 weeks. Single administration of forimtamig reduced tumor load by 80% at the end of cycle 1 (C1). At the end of cycle 4 (C4), all mice achieved a complete response (CR) with no detectable tumors. However, between C4 and cycle 6 (C6), tumor escape was observed in 47% of animals and progression free survival rate (PFS) at study termination was 53%. Relapsed tumors were subjected to quantitative immunohistochemistry and while prevalence for BCMA was comparable to baseline, GPRC5D expression was lost. To evaluate if cereblon modulation represents a strategy to improve PFS, we combined fixed-duration forimtamig with either pomalidomide (pom) or iberdomide (iber) at C1 day 1 (C1D1). Combination with pom and iber increased anti-tumor response compared to monotherapy during C1 deleting 88% and 92% of tumors, respectively. PFS for the combination with pom was not improved (~50%) as tumor escape was observed after treatment was stopped at C6. In contrast, no tumor relapse was observed for the combination with iber increasing PFS to 100%. Notably, as compared to forimtamig monotherapy, combination with pom and iber further increased serum levels of IFNg, IP-10, IL-2 and TNFa 48h after TCB dosing. We next explored if a sequential start of the combination would impact PFS and cytokine release and started co-administration at C3 instead of C1. Forimtamig induced strong tumor regressions in 90% of animals during C1-C4, but tumor escape was observed in 100% of animals starting after C4. When combined with pom or iber, PFS rates increased to 30% and 60%, respectively. Overall cytokine release was significantly lower compared to C1 and neither addition of pom nor iber led to an increase in cytokine production as compared to forimtamig monotherapy. To explore the potential of CELMoD combination at significantly lower TCB starting and target doses, we introduced step up dosing for forimtamig reflecting clinically relevant exposures and combined with thrice weekly (Q3W) or five time a week (Q5W) dosing of mezigdomide (mezi) starting at C1D1. Although forimtamig induced transient tumor regressions at C1D1 as well C1D15, mice showed progressive disease at the end of C1 and only 20% of mice achieved a CR at the end of the study. In contrast, combination with mezi led to rapid onset of tumor regression during C1 correlating with significantly improved PFS rates of 80% (Q3W) and 100% (Q5W). Cytokine release was measured after each step up and target dose and a maximal 2-fold increase in combination with mezi was detected. Interestingly, in contrast to IL-2 or IP-10, serum levels of IL-8 as well as MIP-1a decreased for the Q3W mezi schedule. Taken together, our data suggest that combination with CELMoDs but not IMIDs can prevent relapse to forimtamig driven by GPRC5D negative tumor cells. We confirm timing of intervention with CELMoDs to have an significant impact on PFS and cytokine release and suggest a broad therapeutic window using low dose forimtamig and intermittent dosing of iberdomide or mezigdomide. References: 1Mailankody S, Devlin SM, Landa J, et al. GPRC5D-Targeted CAR T Cells for Myeloma. N Engl J Med. 2022;387(13):1196-1206. doi:10.1056/NEJMoa2209900

Supplementary Data from Preclinical Characterization of a Next-Generation Brain Permeable, Paradox Breaker BRAF Inhibitor

Abstract Background: RG6234 is a GPRC5DxCD3 T-cell engaging bispecific antibody (TCB) that redirects T cells to target and eliminate cells expressing GPRC5D, including malignant plasma cells. RG6234 has a novel 2:1 (GPRC5D:CD3) configuration that confers bivalent binding to GPRC5D and increased T-cell directed killing compared with other molecular formats. RG6234 is initiated with Cycle 1 step-up dosing to mitigate the risk for severe cytokine release syndrome (CRS). Introduction: We performed an in silico evaluation of the dynamics of soluble B-cell maturation antigen (sBCMA), used as a surrogate for tumor burden and informing probability of response, and of the maximum release of IL-8, used as a surrogate for immune activation and informing the probability of CRS, using a QSP model. The model was calibrated with clinical data from 43 relapsed/refractory Multiple Myeloma (R/R MM) patients from the ongoing IV dose escalation study (NCT04557150). The model was set up to: 1) perform patient-specific calibrations and characterize the population with regard to patients’ sensitivity to tumor killing and immune activation and associated heterogeneity; and 2) simulate different dosing regimens in virtual populations and predict their probability of response and CRS. Here we present the model development and calibration results. Methods: The QSP model is a minimal mechanistic model integrating key elements of the Mechanism of Action (MoA) of RG6234. It describes immune activation by RG6234 and resulting MM cell killing. It comprises a system of two ordinary differential equations and 20 parameters, two of which are fitted to longitudinal clinical data (sBCMA and IL-8). Mechanistic assumptions regarding the MM disease and the MoA of RG6234 are represented in the model and supported by clinical or preclinical evidence. Of note, the immune tolerance observed in Cycle 1, indicated by the progressive decrease of cytokine peak levels despite the increased step-up dose level, is captured in the model by limiting the number of activated and proliferating T cells in the tumor microenvironment. Results: The calibrated model shows an accuracy of 92% in recapitulating Partial Response or better and of 78% in recapitulating CRS occurrence after the first step-up dose, demonstrating its appropriateness to address clinically relevant questions. Patient specific model calibrations show that the treated R/R MM population is more heterogeneous with regard to its sensitivity to RG6234-induced MM cell killing than to immune activation. Conclusions: The mechanistic model is able to simulate RG6234-induced T-cell mediated tumor cell killing and can be utilized to predict response and CRS in virtual populations after IV administrations at different dosing regimens. A model validation is planned with data from the expansion cohort of the study. Citation Format: Cristina C. Santini, Emilie Schindler, Jan Attig, Jan Eckmann, Suresh Vatakuti, Francesco Brizzi, Antoine Soubret, Sara Belli. Development of a quantitative systems pharmacology model for clinical dose and schedule optimization of RG6234, a T-cell engaging antibody targeting GPRC5D in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 843.

Background: RG6234 is a GPRC5DxCD3 T-cell engaging bispecific antibody (TCB) that redirects T cells to target and eliminate cells expressing GPRC5D, including malignant plasma cells. RG6234 has a novel 2:1 (GPRC5D:CD3) configuration that confers bivalent binding to GPRC5D and increased T-cell directed killing compared with other molecular formats. RG6234 is initiated with Cycle 1 step-up dosing to mitigate the risk for severe cytokine release syndrome (CRS). Introduction: We performed an in silico evaluation of the dynamics of soluble B-cell maturation antigen (sBCMA), used as a surrogate for tumor burden and informing probability of response, and of the maximum release of IL-8, used as a surrogate for immune activation and informing the probability of CRS, using a QSP model. The model was calibrated with clinical data from 43 relapsed/refractory Multiple Myeloma (R/R MM) patients from the ongoing IV dose escalation study (NCT04557150). The model was set up to: 1) perform patient-specific calibrations and characterize the population with regard to patients’ sensitivity to tumor killing and immune activation and associated heterogeneity; and 2) simulate different dosing regimens in virtual populations and predict their probability of response and CRS. Here we present the model development and calibration results. Methods: The QSP model is a minimal mechanistic model integrating key elements of the Mechanism of Action (MoA) of RG6234. It describes immune activation by RG6234 and resulting MM cell killing. It comprises a system of two ordinary differential equations and 20 parameters, two of which are fitted to longitudinal clinical data (sBCMA and IL-8). Mechanistic assumptions regarding the MM disease and the MoA of RG6234 are represented in the model and supported by clinical or preclinical evidence. Of note, the immune tolerance observed in Cycle 1, indicated by the progressive decrease of cytokine peak levels despite the increased step-up dose level, is captured in the model by limiting the number of activated and proliferating T cells in the tumor microenvironment. Results: The calibrated model shows an accuracy of 92% in recapitulating Partial Response or better and of 78% in recapitulating CRS occurrence after the first step-up dose, demonstrating its appropriateness to address clinically relevant questions. Patient specific model calibrations show that the treated R/R MM population is more heterogeneous with regard to its sensitivity to RG6234-induced MM cell killing than to immune activation. Conclusions: The mechanistic model is able to simulate RG6234-induced T-cell mediated tumor cell killing and can be utilized to predict response and CRS in virtual populations after IV administrations at different dosing regimens. A model validation is planned with data from the expansion cohort of the study. Citation Format: Cristina C. Santini, Emilie Schindler, Jan Attig, Jan Eckmann, Suresh Vatakuti, Francesco Brizzi, Antoine Soubret, Sara Belli. Development of a quantitative systems pharmacology model for clinical dose and schedule optimization of RG6234, a T-cell engaging antibody targeting GPRC5D in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 843.

Background: Despite advances in treatment, almost all pts with newly diagnosed multiple myeloma (MM) eventually relapse and progression-free survival decreases with each subsequent line of therapy. New treatments that target novel antigens and/or have novel mechanisms of action (MOAs) are needed. GPRC5D is a G-protein coupled receptor that is overexpressed on malignant plasma cells. Expression on normal tissue is limited to skin (hair follicles and eccrine glands) and testis (seminiferous tubules). In a Phase I study (NCT04557150), RG6234, a GPRC5DxCD3 T-cell engaging bispecific antibody with a novel 2:1 configuration, was highly active in pts with RRMM and had a safety profile consistent with its MOA and target distribution when given IV (Hasselbalch Riley et al. EHA 2022). We report updated IV and first SC results. Methods: All pts had RRMM for which no established therapy was available, appropriate or tolerable and had received ≥1 prior IMiD and ≥1 prior PI. Prior CAR T-cells, antibody-drug conjugates and bispecific antibodies were allowed. RG6234 was initiated with step-up dosing, reaching the target dose 2 weeks after the initial step dose, and was given for up to 1 year unless disease progression or unacceptable toxicity occurred. All pts who received RG6234 in the dose-escalation phase of the study were included in the analysis (IV dose range: 6-10000µg; SC dose range: 30-7200µg). Results: At data cut-off (June 8, 2022), 51 pts had been enrolled into the IV cohorts (median age: 62 years; min-max: 27-78) and 54 into the SC cohorts (64 years; 46-79). Median number of prior lines was 5 (min-max: 2-15) and 4 (2-14), respectively. Many pts were triple-class refractory (IV: 63.3%; SC: 73.1%), some were penta-class refractory (IV: 30.6%; SC: 42.3%), and some had received prior anti-BCMA therapies (IV: 19.6%; SC: 20.4%). High-risk cytogenetics (t(4;14), t(14:16), del(17p)) were common (IV: 46.7% of 30 evaluable pts; SC: 50.0% of 28). Across all tested doses, cytokine release syndrome (CRS) was the most common adverse event (AE; IV: 82.4% of pts; SC: 77.8%). Grade (Gr) ≥3 CRS was uncommon (IV: 2.0%; SC: 1.9%) and most events were confined to Cycle 1. CRS management often involved tocilizumab (IV: 39.2% of pts; SC: 25.9%) and corticosteroids (IV: 52.9%; SC: 25.9%). ICANS-like AEs were infrequent and mostly mild (any Gr: 9 pts [8.6%]; Gr ≥3: 2 pts [1.9%]). AEs related to on-target, off-tumor effects included dermal and epidermal conditions (IV: 72.5%; Gr 3: 11.8%; SC: 81.5%; Gr 3: 14.8%), hair and nail changes (IV: 17.6% [all Gr 1-2]; SC: 22.2% [all Gr 1-2]), and AEs affecting the gastrointestinal mucosal epithelium or tongue (IV: 70.6% [all Gr 1-2]; SC: 74.1%; Gr 3: 5.6%). Gr ≥3 hematologic AEs were infrequent (anemia: IV 13.7%, SC 5.2%; thrombocytopenia: IV 13.8%, SC 18.5%; neutropenia: IV 11.8%, SC 16.7%). Infections were common (IV: 56.9%; Gr ≥3: 19.6%; SC: 37.0%; Gr ≥3: 24.1%). RG6234-related AEs leading to treatment discontinuation occurred in 2 pts (3.9%) in the IV group and 2 pts (3.7%) in the SC group. One RG6234-related Gr 5 (fatal) AE (1.9%) of acute respiratory failure was reported in the SC group. Median follow-up among efficacy evaluable pts in the IV group (7.1 months; min-max: 0.5-16.8; n=49 pts) was longer than in the SC group (3.9 months; min-max: 1.1-10.5; n=48 pts). Overall response rates were 71.4% and 60.4%, respectively (Figure). Responses were observed in 10/18 pts (55.6%) overall who had received prior anti-BCMA therapies and 18/28 pts (64.2%) who had high-risk cytogenetics. Median time to first response in the IV and SC cohorts was 1.4 months (95% CI: 1.2-1.8) and 1.6 months (1.2-2.1), respectively. Duration of response (DoR) data were immature at cut-off. Responses were ongoing in 24/35 pts (68.6%) in the IV cohorts and in 26/29 (89.7%) in the SC cohorts, with a maximum DoR of 12.9 months and 8.8 months, respectively. Biomarker data demonstrated rapid T-cell activation and T cell-mediated anti-MM activity irrespective of administration route (Dekhtiarenko et al. ASH 2022). Anti-drug antibody incidence was low (IV: 7.8%; SC: 1.8%), and impacted RG6234 PK and efficacy in one pt (1.0%) only. Conclusions: RG6234 is highly active in pts with heavily pretreated RRMM when administered IV or SC. AEs are consistent with its MOA and target distribution. Evaluation and optimization of IV and SC dosing is ongoing. Updated data will be presented. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: RG6234 is a GPRC5DxCD3 T-cell engaging bispecific antibody with a novel 2:1 format. GPRC5D is overexpressed on multiple myeloma (MM) cells and concurrent binding of RG6234 to GPRC5D on tumor cells and CD3 on T cells results in immunological synapse formation and potent T-cell directed tumor cell killing (Eckmann et al. ASH 2022). An ongoing Phase I study (NCT04557150) is investigating the safety, clinical activity, pharmacodynamics (PD), and pharmacokinetics of intravenous (IV) and subcutaneous (SC) administration of RG6234 in patients with RRMM. Clinical activity was observed in both dose escalations (overall response rate [ORR] IV: 71.4%; ORR SC: 60.4%) (Carlo-Stella et al. ASH 2022). Here, we report clinical biomarker data comparing the PD effects of IV and SC administration and confirming the mechanism of action and potency of RG6234 when given by both administration routes. Methods: Exploratory biomarker analyses included dose-escalation data from 50 patients who received IV doses ranging from 6-10000μg and 46 patients who received SC doses ranging from 30-7200μg. In both cohorts, RG6234 administration was initiated with step-up dosing, reaching the target dose 2 weeks after the initial step dose. Peripheral biomarkers were evaluated using whole blood flow cytometry (IV, n=33; SC, n=40), plasma cytokines (IV, n=50; SC, n=46) and sBCMA (IV, n=36; SC, n=27) Protein Simple ELLA. MM cells were assessed at baseline and on-treatment by bone marrow (BM) aspirate flow cytometry and by BM biopsy CD138/CD8 immunohistochemistry. The data cut-off for the current analysis was June 8, 2022. Updated data will be presented. Results: RG6234 induced rapid PD changes at all tested doses. With IV administration, cytokines (IFNγ, TNFa, CXCL10, IL6, IL10, IL2, IL8) peaked at 4 to 24 hours after the first infusion and cytokine peaks diminished significantly with subsequent administrations. SC administration resulted in a delayed cytokine release that peaked at 24 to 72 hours after the first injection and was 2.2-24.2-fold lower than with IV dosing. Cytokine peaks were comparably low after each SC step-up administration. After SC dosing, T-cell margination was delayed by 20 hours and recovery to 50% of baseline levels took on average 56 hours longer than with IV dosing. Analysis of paired baseline and on-treatment BM biopsies (IV, n=19; SC, n=8) revealed a comparable degree of T-cell recruitment towards the tumor with IV and SC dosing (2.3- and 1.6-fold increase, respectively, in CD8 T-cell density in responders at the end of Cycle 1). Both IV and SC administration induced rapid depletion of MM cells in responding patients, as demonstrated by a decrease in plasma sBCMA 8 days after the initial administration (median 24.9% [n=27] and 26.2% [n=18] reduction from baseline, respectively). By Cycle 3 Day 1, median decreases from baseline in plasma sBCMA among responders were 91.8% (n=24) after IV administration and 95.6% (n=12) after SC administration. Moreover, at the end of Cycle 1, the majority of patients (15/16 [93.8%] in the IV group and 14/15 [93.3%] in the SC group) had <1% MM cells in BM based on flow cytometry. GPRC5D expression was detected at baseline in almost all patients with evaluable BM aspirate (44/45, median of 94.7% GPRC5D+ MM cells). Conclusions: Biomarker analysis indicates that RG6234 leads to T-cell engagement in the BM of patients with RRMM and demonstrates rapid and effective T-cell mediated anti-myeloma activity irrespective of the route of administration. Cytokine release, T-cell activation followed by BM infiltration, and MM cell depletion are early PD changes after IV and SC administration and precede clinical responses. Compared with IV dosing, SC administration reduces peak cytokine levels in Cycle 1, suggesting a more subtle immune activation and a potential safety benefit for patients.

With the majority of patients relapsing after multiple lines of diverse treatments, multiple myeloma (MM) remains a largely incurable disease. "Off the shelf" T-cell bispecific antibodies (TCBs) targeting GPRC5D demonstrated promising efficacy in early clinical development. Here we benchmarked RG6234, a novel 2:1 GPRC5D-TCB, versus a 1:1 GPRC5D-TCB and a 2:1 BCMA-TCB in clinically relevant ex vivo and in vivo models confirming its' best in class TCB potential for the treatment of multiple myeloma. First we compared the potency of TCBs using MM patients' immune cells and MM cell lines with a wide range of GPRC5D expression levels. RG6234 exhibited superior potency in T cell activation, cytokine production and proliferation against all GPRC5D+ MM cell lines tested. Particularly on target cells with low expression the potency and efficacy of RG6234 was clearly superior to a conventional 1:1 GPRC5D-TCB. Next we compared the potential of TCBs to drive T cell activation and MM plasma cell (PC) depletion in an autologous ex vivo model of MM using total BM aspirates from newly diagnosed MM patients. RG6234 exhibited superior potency as illustrated by upregulation of CD25 on CD4+ and CD8+ T cells and superior efficacy as demonstrated by lower EC50 MM PC depletion dose of 0.12 nM versus 6.26 nM for 2:1 BCMA-TCB and 41.9 nM for 1:1 GPRC5D-TCB. We next tested the capacity of TCBs to inhibit growth of established NCI-H929 xenograft tumors in humanized mice (huNSG) upon repetitive dosing. Strikingly, RG6234 therapy eradicated tumors at all doses tested (0.1-1-10 mg/kg) whereas no regressions were observed for 1:1 GPRC5D-TCB. Interestingly, despite higher baseline expression of BCMA at treatment start, 2:1 GPRC5D-CD3 TCB was also clearly more efficacious than the 2:1 BCMA-CD3 TCB underlying the best in class TCB potential for treatment of multiple myeloma. To improve mechanistic understanding of the mode of action of RG6234 in a patient relevant setting we next evaluated T cell recruitment, activation and MM PC killing in an orthotopic in vivo model of multiple myeloma in huNSG mice. RG6234 was highly active eliminating multiple myeloma cells in the BM as early as 72h after single dose injection as confirmed by drop in soluble BCMA. GPRC5D-TCB induced CD8 T cell margination in blood 24h after first injection and T cell expansion at 24h after second dosing. Up to 5 fold T cell expansion was observed in BM tumors 72h after first and second dosing indicating efficient cross-linking of T cells at the tumor site. Timing of T cell expansion and tumor cell killing correlated with shift of naïve CD62L+CD45RA+ towards CD4RA-CD62L- effector memory CD8 T cells in blood and in tumor. In line with the proposed TCB mode of action, RG6234 induced release of patient relevant amounts of cytokines immediately after first but not after second dosing, correlating with tumor burden at the given timepoints. Peak concentrations for individual cytokine were detected at 4h (IL-2, MIP1a and GM-CSF), 24h (TNFa, CXCL10, G-CSF and IL-10) and 48h-72h ( INFg, IL-6, IL-8 or sCD25). We next evaluated the therapeutic capacity of RG6234 in combination with daratumumab (Dara) and/or pomalidomide (Pom) in total BM aspirate samples from 10 MM patients with variable frequency of GPRC5D positive MM plasma cells (range 74-98%). RG6234 induced MM PC lysis was seen in all patients and was significantly boosted in combination with Dara or the triple combination with Dara and Pom. Tumor cell killing was correlated with increased expression of CD69, CD25, and CD107a as well as checkpoints CD137 and PD-1 on CD8 T cells. Only combination with Pom but not Dara enhanced T cell activation and cytokine release underpinning the different modes of action of these standard of care agents. To validate synergistic combination activity upon repetitive dosing huNSG mice were engrafted s.c. with RPMI-8226 or OMP-2 tumors and treated with RG6234 in combination with Dara or Lenalidomide (Len), respectively. Antitumoral response of RG6234 was significantly improved in combination inducing tumor stasis with Dara and regressions with Len. Improved efficacy in combination with Len was found to correlate with significant expansion of intratumoral T cells 48h after second injection. In summary, we demonstrate preclinical evidence for best in class potential of the RG6234 antibody format for the treatment of multiple myeloma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background Acute Myeloid leukemia (AML) is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. CD25 Mab (also referred to as RG6292 and RO7296682) is an afucosylated, IL-2 non-blocking human IgG1 antibody, shown to efficiently deplete immunosuppressive regulatory T cells (Tregs) in humans and solid tumour models (Kolben 2021) whilst allowing binding of IL-2 to effector T cells and the induction of anti-tumour adaptive immune responses (Solomon, Amann et al. 2020). CD25 Mab binds to CD25+ target cells and its crystallisable fragment (Fc) to Fc receptors (FcR) expressed on the surface of effector cells, such as FcgRIIIa on Natural Killer (NK) cells, monocytes and macrophages. It mediates killing of target cells through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). CD25 Mab is currently being investigated in a phase I dose escalation monotherapy study (NCT04158583) or in combination with atezolizumab (NCT04642365). In AML patients, a higher frequency of immunosuppressive bone marrow Tregs was observed as compared to that of healthy donors and their abundance correlated with poorer outcome (Szczepanski, Szajnik et al. 2009, Han, Dong et al. 2018, Dong, Han et al. 2020). In addition, CD25 is also expressed on a subset of AML cells and may be restricted to leukemic stem cells (LSC) or cells with a progenitor phenotype (Angelini, Ottone et al. 2015, Bertolini, Kageyama et al. 2018, Aref, Azmy et al. 2020). Considering the critical role of LSC in the propagation of the disease and the high relapse rate in AML, it would be important to understand if CD25 targeting could deepen responses and help prevent relapse in patients. In this study, we hypothesized that CD25 Mab may have a dual mode of action whereby it depletes suppressive Tregs and has a direct cytotoxic effect on CD25+ AML cells. Methods We conducted an in-depth characterization of bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry (up to 39 cellular markers) and RNA sequencing using computational analysis. Moreover, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab on Tregs and CD25+ AML cells, as compared to isotype control antibody. Results We found that the abundance of Tregs and CD25+ AML cells correlated strongly with that of the blood in 11 patients with time-matched samples, indicating that blood samples could be used for the identification of predictive biomarkers. Moreover, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing internal tandem duplication mutations in the FLT3 gene (FLT3-ITD). Furthermore, all four patients treated with a hypomethylating agent in combination with venetoclax displayed detectable levels of CD25+ AML cells. We adopted a patient-centric approach (n=14 patients) to study AML clusters with CD25 expression, due to the high inter-patient heterogeneity. Interestingly, CD25+ clusters most commonly expressed CD34, an LSC marker indicating a preferential expression in immature AML cells. Finally, ex vivo treatment of primary AML patient samples with CD25 Mab led to the specific killing of CD25+ AML cells and Tregs by allogeneic NK cells, as compared to isotype control treatment. Taken together, these results provide a proof of concept of CD25 Mab's dual mode of action in AML patient samples. Conclusions Using high dimensional flow cytometry and computational analysis, we provide a deep characterization of AML patient samples and highlight the heterogeneity in the pattern of expression of common AML targets currently under clinical investigation. We dissected the expression of CD25 on healthy T cells and malignant cells and demonstrated that the blood offers a window into the bone marrow composition and could be used to ascertain Treg and CD25+ AML cells prevalence. Mode of action studies demonstrated that CD25 Mab depletes suppressive Tregs and has a direct cytotoxic effect on the CD25+ AML cells. CD25 targeting represents an attractive target for the treatment of AML, especially in patients where CD25 is expressed on LSC or immature AML cells. Our study warrants further exploration of CD25 Mab as combinatorial treatment with, for instance, FLT3 inhibitors or venetoclax.

Abstract Purpose: Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi. Experimental Design: The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi. Results: Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples. Conclusions: The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation.

Abstract The therapeutic benefit of approved BRAF and MEK inhibitors (BRAFi/MEKi) in patients with brain metastatic BRAF V600E/K–mutated melanoma is limited and transient. Resistance largely occurs through the restoration of MAPK signaling via paradoxical BRAF activation, highlighting the need for more effective therapeutic options. Aiming to address this clinical challenge, we characterized the activity of a potent, brain-penetrant paradox breaker BRAFi (compound 1a, C1a) as first-line therapy and following progression upon treatment with approved BRAFi and BRAFi/MEKi therapies. C1a activity was evaluated in vitro and in vivo in melanoma cell lines and patient-derived models of BRAF V600E–mutant melanoma brain metastases following relapse after treatment with BRAFi/MEKi. C1a showed superior efficacy compared with approved BRAFi in both subcutaneous and brain metastatic models. Importantly, C1a manifested potent and prolonged antitumor activity even in models that progressed on BRAFi/MEKi treatment. Analysis of mechanisms of resistance to C1a revealed MAPK reactivation under drug treatment as the predominant resistance-driving event in both subcutaneous and intracranial tumors. Specifically, BRAF kinase domain duplication was identified as a frequently occurring driver of resistance to C1a. Combination therapies of C1a and anti–PD-1 antibody proved to significantly reduce disease recurrence. Collectively, these preclinical studies validate the outstanding antitumor activity of C1a in brain metastasis, support clinical investigation of this agent in patients pretreated with BRAFi/MEKi, unveil genetic drivers of tumor escape from C1a, and identify a combinatorial treatment that achieves long-lasting responses. Significance: A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.