Immune Modulation in Solid Tumors: A Phase 1b Study of 2 RO6870810 (BET Inhibitor) and Atezolizumab (PD-L1 Inhibitor) 3

ABSTRACT


BACKGROUND
Epigenetic modifications are fundamental in guiding gene expression patterns, and alterations in these modifications are frequently associated with the onset of various malignancies (1).One prominent mechanism of epigenetic regulation is the reversible acetylation of histones, which allows for dynamic gene expression modulation in response to various stimuli.At the heart of this process is the Bromodomain and extra-terminal domain (BET) protein family, which includes BRD2, BRD3, and BRD4, and the testis-specific BRDT.Serving as epigenetic "readers", these proteins specifically identify and bind to acetylated histones (2).
Recently, the therapeutic promise of BET protein inhibition has emerged, leading to the development of small molecule BET inhibitors (BETi), such as JQ1, which acts by binding the bromodomains of BET proteins, inhibiting their chromatin association and thereby modulating gene expression (3,4).RO6870810 (also known as RG6146 or TEN-010), is a new non-covalent BETi.Despite its structural resemblance to JQ1, RO6870810 has superior solubility and stability, potentially offering therapeutic advantages (5).BRD4, a primary target of RO6870810, is a universal gene transcription regulator (6).It has been linked to the upregulation of oncogenes like MYC, BCL2, CDK6, and FOSL1 (7)(8)(9)(10).Notably, BRD4 preferentially binds to super-enhancers, which are vast regulatory regions known for controlling genes necessitating high expression levels (11,12).While the sensitivity to BETi isn't solely dictated by super-enhancers (6,13), genes adjacent to these regions may be linked to BRD4 inhibition.
. In hematological malignancies, particularly those with MYC and BCL2 overexpression due to super-enhancer-driven transcriptional control, BETi has shown moderate success (14,15).
Accumulating evidence also suggests potential BETi susceptibility in solid tumors, like triplenegative breast cancer (TNBC) and advanced ovarian cancer which presently need effective treatments.Notably, BRD4 amplification has been documented in these cancers (16), and MYC amplification is prevalent in recurring ovarian tumors (17).Furthermore, BET proteins' roles in immune function have potential utility in cancer therapy.While early research highlighted JQ1's ability to suppress immune regulators in various tumor models (18)(19)(20), newer preclinical studies showcase BETi's diverse impacts on immune cell subtypes and activation.
The safety and efficacy of the BET inhibitor RO6870810 combined with venetoclax and rituximab was previously investigated for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL) (20).In this phase 1b trial involving 39 patients, the combination therapy showed tolerability with manageable toxicities.Dose-limiting toxicities included neutropenia, diarrhea, and hyperbilirubinemia.The maximum tolerated dose (MTD) for the combination of RO6870810 and venetoclax was established at 0.65 mg/kg for RO6870810 and 600 mg for venetoclax.For the triple combination of RO6870810, venetoclax, and rituximab, the MTDs were 0.45 mg/kg, 600 mg, and 375 mg/m², respectively.The combination showed promising anti-tumor activity with an overall response rate of 38.5% and complete responses in 20.5% of patients.
Another phase 1b trial was conducted to determine the maximum tolerated dose (MTD) and optimal biological dose (OBD) of RO6870810 monotherapy in patients with advanced multiple myeloma (21).Though pharmacodynamic results indicated the on-target effects of RO6870810, clinical responses were infrequent and, when present, transient.These findings align with the .preliminary activity noted for RO6870810 in an earlier first-in-human dose-escalation study.
In a study by Roboz G.J. et al. (22), 32 patients with relapsed/refractory acute myeloid leukemia and hypomethylating agent-refractory myelodysplastic syndrome were treated with RO6870810 monotherapy (22).Significant reductions in circulating CD11b+ cells, a known pharmacodynamic marker of BET inhibition, were observed at RO6870810 concentrations exceeding 120 ng/mL.Most side effects were mild, and there were no treatment-related fatalities.Although some patients showed signs of stabilization or remission, the development of RO6870810 as a standalone therapy was discontinued due to its limited efficacy.
The ability of BETi to inhibit the PD-1/PD-L1 immune checkpoint pathway and bolster anti-tumor immunity suggests that combining it with a checkpoint inhibitor could yield improved clinical outcomes (23).Supporting this notion, preclinical studies using a combination of BETi with anti-PD-1 or anti-PD-L1 antibodies have showcased synergistic anti-tumor effects in mouse models of lymphoma (19), melanoma (24), and non-small cell lung cancer (25).Yet, clinical evidence from such combination therapies remains unreported (23,26).
In this study, we present findings from a phase 1b clinical trial involving TNBC and ovarian cancer patients.These patients received treatment with the BETi RO6870810 as a monotherapy or in combination with atezolizumab (Tecentriq), a humanized IgG1 monoclonal antibody targeting PD-L1.Notably, atezolizumab has secured approval for treating PD-L1 positive metastatic .TNBC (27).Our study examines the potential anti-tumor immune activation facilitated by both RO6870810 monotherapy and its combination with atezolizumab.We offer a detailed biomarker analysis, highlighting transcriptional alterations and immune modulation in both tumor tissue and peripheral blood.This is the first study to explore the effects of combining BET inhibition with PD-L1 blockade to enhance therapeutic efficacy by targeting both the epigenetic regulation pathways and immune checkpoint pathways simultaneously.

Study design
We conducted a phase 1b, open-label, non-randomized trial on patients ≥18 years with TNBC and advanced ovarian cancer.We explored two treatment strategies: (1) immediate combination of RO6870810 administered subcutaneously, with intravenous atezolizumab (concomitant regimen, Fig. 1a), and (2) an initial 21-day single-agent, subcutaneous RO6870810 treatment, followed by its combination with intravenous atezolizumab (sequential regimen, Fig. 1b).The dose-escalation followed a classic 3+3 design with initially planned doses of 0.30 mg/kg, 0.45 mg/kg, and 0.65 mg/kg.The study had four groups.Groups 1 and 2 focused on dose escalation for the concomitant and sequential treatments, respectively.Patients in group 1 received a starting-dose of 0.30 mg/kg for 14 days administered subcutaneously on a 3-week schedule.Once a cohort in group 1 was completed and deemed safe, group 2 began the 21-day run-in period, during which RO6870810 monotherapy was administered to a minimum of 3 participants.Participants enrolled in group 2 initially received RO6870810 as monotherapy during the first 14 days of a 21-day run-in period, starting at a dose of 0.30 mg/kg.Patients in the same dose level were treated simultaneously.
Following the run-in period, participants continued to receive RO6870810 at the same dose in combination with 1200 mg atezolizumab in 21-day cycles.In the expansion phase, Cohorts 3 and 4 further investigated the concomitant regime for TNBC and ovarian cancer patients, using the optimal dose determined in Cohort 1.
The study primarily aimed to ascertain the maximum tolerated dose (MTD) or maximum administered dose (MDA) of RO6870810 both as a standalone treatment and in combination with atezolizumab, by monitoring dose-limiting side effects and ongoing safety.The expansion groups enabled us to gauge the early clinical efficacy of RO6870810 when paired with atezolizumab.
Additionally, understanding the immune modulation profiles of RO6870810, both as monotherapy and when combined with PD-L1 inhibition, was a goal for this study.The study's methodology, eligibility criteria, dosing schedules, and safety protocols are detailed in the Supplementary Methods.Further information is accessible on ClinicalTrials.govunder trial ID NCT03292172 or via this direct link: https://clinicaltrials.gov/study/NCT03292172.

Sample collection and analysis
Blood samples were collected at specified intervals for biomarker analysis.Flow cytometry was conducted at Covance Central Laboratory using established protocols.Cytokine levels were measured using the ELLA method by Microcoat Biotechnologie.Tumor biopsies were processed for immunohistochemistry and RNA-seq to study gene expression and pathway activity.The .detailed methods, including sample preparation, analytical procedures, and statistical analyses, are provided in the supplementary methods section.

Dosing of RO6870810
The dosing rationale was based on pharmacokinetic profile and tolerability of RO6870810 observed in patients with NUT carcinoma, other solid tumors, and DLBCL (21).In this study, RO6870810 demonstrated overall tolerability across different indications except for a single doselimiting toxicity (DLT) of grade 3 cholestatic hepatitis observed in a patient with prostate cancer at 0.45 mg/kg on a 28-day schedule.This led to the expansion of the cohort without additional DLTs and dose escalation to 0.65 mg/kg.Although no DLTs were reported at this level during cycle 1, treatment discontinuations due to fatigue in cycle 2 prompted the exploration of a 14 of 21 days schedule.This 0.65 mg/kg dose was identified as the recommended phase 2 dose for solid tumors.Similarly, in the study by Dickinson et al.(15), the maximum tolerated dose (MTD) for the combination of RO6870810 and venetoclax was established at 0.65 mg/kg for RO6870810 and 600 mg for venetoclax.For the triple combination of RO6870810, venetoclax, and rituximab, the MTDs were determined to be 0.45 mg/kg for RO6870810, 600 mg for venetoclax, and 375 mg/m² for rituximab.
Based on the safety profile and pharmacodynamic (PD) effects observed, a starting dose of 0.3 mg/kg for 14 days on a 3-week schedule was selected as appropriate for the initial dose cohort of both groups.This dosage was anticipated to provide significant target PD effects while maintaining a tolerable safety profile.This strategy aimed to optimize the therapeutic potential of RO6870810 in combination with atezolizumab for the patient population in this study.

Participants
Thirty-six (36) patients with metastatic advanced ovarian cancer (n= 29) or triple negative breast cancer (n= 7) were included and received at least one dose of study drug in this open-label, dose finding and expansion phase 1 study.The total of 36 safety evaluable patients were enrolled in Denmark (8 patients), Canada (10 patients), the US (15 patients), and Australia (3 patients).Details of the groups and cohorts and their dosages are provided in Table 1.
Twenty-seven patients were included in the dose escalation part (groups 1 and 2) and 9 patients were treated in the expansion phase at the recommended phase 2 dose of 0.

Safety
The study was terminated prematurely because of frequency and severity of adverse events (AEs) and an unfavorable risk-benefit profile of the combination of RO6870810 and Atezolizumab.The study recorded 15 deaths (41.7%), with nine deaths due to progressive disease and six deaths (16.7%) reported during long-term follow-up where the cause of death was unknown.None of the deaths were treatment-related.
Although there were laboratory abnormalities in both hematological (high and low) and clinical chemistry (high and low) parameters, none of these were considered clinically significant.No clinically meaningful differences from baseline were noted in the vital signs.

Efficacy
Response was measured according to RECIST overall response.Out of 31 evaluable patients, two patients exhibited a partial response (PR), fifteen patients demonstrated stable disease (SD), and fourteen patients were classified with progressive disease (PD) as their best objective response (Figure 2).Further breakdown and detailed analysis of patient responses across different groups and cohorts are documented in Table 2.
The two partial responses were observed in Group 1, Cohort 1, which received a dosage of 0.3 mg/kg concurrently, and in Group 1, Cohort 2, with a 0.45 mg/kg concurrent dosage.Five patients were excluded from the clinical response evaluation due to the absence of post-baseline response data and were therefore categorized as having progressive disease.

Pharmacodynamic effects for BETi biomarkers
Pharmacodynamic (PD) biomarkers for RO6870810 were evaluated in peripheral blood and tumor tissue.BET inhibitors are known to target peripheral blood monocytes (28), which are critical determinants of cancer-associated inflammation.A previous study with RO6870810 has suggested that circulating monocyte levels in peripheral blood can be used as a potential biomarker for the pharmacodynamic effects (29).Figure 3A presents the results of circulating monocytes following concurrent or sequential administration of RO6870810 and atezolizumab.We observed a significant decrease in CD14+/CD11b+ monocytes after the initial treatment cycle, with the lowest counts recorded between days 8 and 14 post-treatment.These levels then recovered by day 21.
We further investigated the expression of genes affected by BET inhibitors (BETi) within the tumor tissue using RNA sequencing (RNA-seq).The genes CD180, CCR2, MYC and HEXIM1 are previously reported pharmacodynamic markers of BETi in different settings (26).On day 21, significant reductions in the levels of CCR2 and CD180 were confirmed under both the concurrent regimen and the monotherapy initiation with RO6870810, while MYC and HEXIM1 were not significantly affected (Figure 3B).The treatment also led to the downregulation of the BRD4 super enhancer, alongside specific changes in the expression of apoptotic and BCL2 family genes (Figure 3C).Notably, BCL2 and BCL2L1 were upregulated, whereas IGLL5 and IRF4 were downregulated.These gene expression changes, particularly within the context of apoptosis and lymphocyte regulation, underscore the potential mechanisms through which RO6870810 exerts its anti-tumor effects.
We also examined the changes in cellular subsets and soluble biomarkers within peripheral blood as assessed by flow cytometry and cytokine profiling.Besides the decrease in CD14+/CD11b+ .monocytes discussed above, no notable changes were observed for the run-in cycle with RO6870810 alone.In contrast, early phases of the combination therapy with atezolizumab were characterized by a transient reduction in circulating immune cells, including CD4+ and CD8+ cells, CD16+CD56+ NK cells, CD19+ B cells, and CD14+/CD11b+ monocytes (Figure 4A).The transient drop in circulating immune cells, potentially due to margination and extravasation, has been previously described for other immunotherapeutic modalities involving T cell activation (30,31).Following this initial reduction in circulating immune cells, there was an expansion of specific cell types, particularly CD16+CD56+ NK cells and CD8+ T cells, but not CD4+ T cells (Figure 4A).Consequently, the ratio of CD4+ to CD8+ T cells shifted towards a higher proportion of cytotoxic cells in the later phase of the combination therapy (Figure 4B).
In the combination therapy with atezolizumab, the concentration of sCD25, a soluble form of the IL-2 receptor alpha chain, showed a marked increase on day 15 post-treatment initiation, with levels remaining elevated through day 21 (Figure 4C).This elevation in sCD25 is indicative of T cell activation, suggesting enhanced immune activation potentially conducive to antitumor activity.Similarly, TNFα, a critical cytokine in inflammation and immune regulation, exhibited a marked increase on-treatment with a peak at day 15 (Figure 4D).These effects were not observed during the run-in cycle with RO6870810 alone, suggesting that the immune-stimulating effects in the combination therapy are driven by atezolizumab.
We subsequently examined tumor tissue by RNA-seq in order to explore immune gene and signature expression changes (Figure 5).Consistent with the established mechanism of action of the PD-L1 inhibitor atezolizumab, we confirm up-regulation of immune effector gene signatures in tumor tissues under the combination therapy, including signatures associated with CD8+ T cell Despite the promising preclinical evidence suggesting potential synergistic effects of combining BET inhibitors with checkpoint inhibitors, our phase 1b study highlights significant challenges and limitations associated with this therapeutic strategy.
Although each agent has a manageable safety profile when used alone, the combination of RO6870810 and atezolizumab led to pronounced immune-related adverse events (irAEs), necessitating premature study termination.The majority of patients experienced treatment-related adverse events, with a substantial proportion encountering severe (Grade ≥3) adverse events and serious adverse events (SAEs).Notably, systemic immune activation (SIA) was a prominent SAE, underscoring the potential for heightened immune responses when combining these agents.These findings align with the known immune-stimulatory effects of checkpoint inhibitors but suggest that the addition of BET inhibition may exacerbate these responses, leading to an unfavorable riskbenefit profile.
Pharmacodynamic analyses confirmed target engagement by RO6870810, as evidenced by changes in established BETi biomarkers in both peripheral blood and tumor tissue.However, contrary to preclinical expectations, RO6870810 monotherapy did not significantly decrease tumor PD-L1 expression and appeared to suppress anti-tumor immunity within the tumor microenvironment (TME).This immunosuppressive effect was only reversed when RO6870810 was combined with atezolizumab, which induced immune effector activation in the TME.This highlights the pivotal role of atezolizumab in stimulating anti-tumor immunity, consistent with its known mechanism of action as a PD-L1 inhibitor.
. The combination therapy also induced systemic immune effects, evidenced by transient reductions in circulating immune cells followed by their expansion, and increased levels of soluble immune activation markers such as sCD25 and TNFα.These systemic changes suggest that while the combination can activate the immune system, it may also predispose patients to severe irAEs.
The observed changes in both circulating immune cells and soluble factors, following concomitant and sequential administration of the treatments, but not with the monotherapy run-in phase using RO6870810 alone, underscore the critical role of atezolizumab in eliciting the potential antitumor immune response.Atezolizumab, by enhancing immune activation and possibly improving the recognition and elimination of tumor cells, emerges as the primary driver behind the immune modulatory effects observed, rather than RO6870810.
The anti-tumor activity observed in this study was limited, with only two patients (5.6%) achieving partial responses.This modest efficacy, coupled with the high incidence of severe irAEs, further supports the conclusion that the combination of RO6870810 and atezolizumab does not provide a favorable therapeutic benefit for patients with advanced ovarian carcinomas and TNBC.The lack of significant tumor PD-L1 modulation by RO6870810 and the observed immunosuppressive effects during monotherapy suggest that BET inhibition may not enhance the efficacy of checkpoint inhibitors in these cancer types.
Our study underscores the complexity of combining epigenetic modulators with immunotherapies.
The anticipated synergistic stimulation of anti-tumor immunity from combining BET inhibitors with checkpoint blockade, as suggested by preclinical models, could not be confirmed in our clinical study.This discrepancy underscores the significant challenges in translating preclinical .findings to clinical settings.The observed immunosuppressive effects of BETi monotherapy within the TME suggest that we need better models and a deeper understanding of the contextdependent effects of these agents on immune modulation.

CONCLUSIONS
The combination of RO6870810 and atezolizumab demonstrated some immune activation; however, the associated severe irAEs and limited anti-tumor efficacy indicate that this therapeutic approach is not viable for patients with advanced ovarian carcinomas and TNBC.These findings highlight the importance of careful evaluation of combination strategies in clinical trials and the need for continued exploration of novel therapeutic approaches to improve outcomes for patients with these challenging malignancies.A. Enrichment scores for key immune pathways (34).Green indicates significant upregulation in combination therapy, suggesting enhanced immune activity.Purple marks downregulation in BETi monotherapy, implying reduced immune response.

B.
Gene expression changes related to CD8 T effector, immune checkpoint, and antigen processing machinery pathways are highlighted.Red represents upregulated genes, reflecting pathway activation, while blue indicates downregulated genes, signifying pathway suppression.Significant changes are marked with asterisk.
.  Response according to RECIST overall response.Responder is defined as any subject who exhibits a complete response or partial response.Missing response is assumed as a non-responder. .
Objective responses were assessed by investigators according to RECIST v1.1 and Immune Modified RECIST criteria.The grading of all adverse events (AEs) was based on the National Cancer Institute Common Toxicity Criteria for Adverse Events (NCI CTCAE) version 4.03.

Figure 1 .
Figure 1.Schematic Overview of Study Treatment Regimens and Pharmacodynamic Biomarker Collection A. The concomitant regimen involved patients receiving a combination of RO6870810 and atezolizumab from initiation.Tumor biopsies for RNA-sequencing and immunohistochemistry (IHC) were taken at baseline (Cycle 1 Day 1 [C1 D1]) and post-first cycle (Cycle 1 Day 21 [C1 D21]), indicated by purple arrows.Peripheral blood samples for flow cytometry and cytokine profiling, shown by red arrows, were collected on days 1, 8, 15, and 21.This regimen was applied to patients in the dose escalation and both expansion cohorts.B. To evaluate the impact of RO6870810 as a single agent, an alternative group followed a sequential regimen, starting with RO6870810 alone in a run-in cycle before transitioning to combined treatment with atezolizumab.Tumor biopsies were performed at the run-in start (Runin Day 1 [RI D1]), post-run-in cycle (Run-In Day 21 [RI D21]), and after the initial cycle of combination therapy (C1 D21).Peripheral blood sampling occurred on the same days during the run-in and the first combination treatment cycle, facilitating a comprehensive analysis of treatment-induced changes.

Figure 2 :
Figure 2: Changes in Target Lesion Size and Best Overall Response Each bar represents the response of an individual patient, measured according to RECIST overall response criteria.The y-axis corresponds to the maximum percentage change from baseline in sum of longest diameters (SLD) in target lesions.Colors indicate the best overall response.Out of 36 patients, 31 were evaluable for clinical response.Two patients who exhibited a decrease in target lesion size were still classified as having progressive disease due to progression in non-target lesions or the appearance of new lesions.

Figure 3 .
Figure 3. Pharmacodynamic Responses of BET Inhibitor Biomarkers in Peripheral Blood and Tumor Tissue A. Quantification of CD14+/CD11b+ monocyte populations in peripheral blood, illustrating changes from baseline (expressed as log2 fold-change from cycle onset) for individual patients (denoted as points), with longitudinal data from the same individual linked.Patients lacking

Figure 4 .
Figure 4. Assessment of Immune Modulation by Flow Cytometry and Cytokine Analyses A. The variation in immune cell populations within peripheral blood, as determined by flow cytometry.Color depicts the log2 fold-change from baseline at each defined time point (refer to Fig. 1 for time points).Red indicates an increase, blue a decrease in cell population frequency, with significant alterations marked by an 'X' (FDR corrected p-value < 0.05).

.B.
Change from baseline in the CD4+/CD8+ cell ratio in peripheral blood, indicating shifts towards either T helper cells (positive values) or cytotoxic cells (negative values).Continuous lines connect sequential time point samples from individual patients, highlighting specific cases of interest in color.Boxplots aggregate data at each time point.C, D. Changes in soluble CD25 (sCD25) and TNFα levels from baseline in peripheral blood.The visualization follows the format of Panel B.

Figure 5 .
Figure 5. Differential Impact of BET inhibitor Monotherapy and Atezolizumab Combination Therapy on Immune Effector Pathways Heatmaps illustrate the contrasting effects of atezolizumab combination therapy and BET inhibitor monotherapy on immune effector pathways within tumor tissues, based on RNA sequencing data.

Table 1 Study Patients, by Group
Expn = Expansion; OC = ovarian cancer; TNBC = triple negative breast cancer