We previously described the capacity of RO7122290 (RO) - a FAP-targeted 4-1BB bispecific antibody - to induce CD8+ T cell infiltration and activation in the tumor (Moreno V. et al, SITC 2020). Aiming to compare pharmacodynamic (PD) changes in tumor nests and stroma, paired tumor biopsies from patients treated with RO (Part A) and RO + atezolizumab (Part B) were analysed by digital spatial profiling (DSP, Nanostring).
Methods
Seven paired (baseline and on-treatment) FFPE tumor tissue biopsies (three from Part A, four from Part B) obtained from an ongoing Phase 1/1b trial (EUDRACT 2017-003961-83) were assessed for mRNA and protein expression. Biopsies were taken from six different tumor types at different RO doses. Up to twelve Regions of Interest (ROIs) were collected per slide and the morphology markers PanCK, CD8, CD3 and DAPI were applied. The ROIs were further annotated in tumor nests and stroma segments based on PanCK staining. The immune-oncology 58-plex protein and 78-plex mRNA expression panels (Nanostring) were used to profile all samples. Data were normalized according to Nanostring guidelines and filtered based on relevance (absolute log2 fold change > 1) and significance (FDR < 0.05, p-value).
Results
The level of CD8+ T cell infiltration measured by spatial profiling correlated with the level measured by IHC, in both tumor nests and stroma. The activation markers 4-1BB and PD-1 were upregulated, confirming the PD effect already measured by mRNA sequencing. We also identified novel protein markers - CD40, PD-L1 and IDO1 - being upregulated after treatment. Spatial regulation differed among the markers with 4-1BB, PD-1 and CD40 upregulated only in the stroma, PD-L1 and IDO1 upregulated in the tumor nests and in the stroma. IDO1 induction is particularly relevant, since this protein is known to attenuate 4-1BB-mediated effector responses. Conventional IHC analysis performed on 14 paired biopsies confirmed IDO1 being upregulated in 11 out of 14 cases and revealed dendritic cells, macrophages and stromal cells to express IDO1. Importantly, IDO1 upregulation was observed in both Part A (3 out of 3) and Part B (8 out of 11).
Conclusions
Spatial profiling allowed us to identify key markers that are spatially regulated after treatment and to gain new insights on the MoA of RO. The induction of IDO1 by RO confirms the dual immunoregulatory nature of 4-1BB signaling and highlights IDO1 as a potential resistance mechanism for RO in the clinical setting, both as single agent and in combination with atezolizumab.
Moreno V. et al, Pharmacodynamic assessment of a novel FAP-targeted 4–1BB agonist, administered as single agent and in combination with atezolizumab to patients with advanced solid tumors, Nov 1 2020, Journal for ImmunoTherapy of Cancer, presented at SITC 2020
<p>Supplementary Table 1 Downregulation of HER3 membrane protein expression in skin biopsies following treatment with lumretuzumab and cetuximab or erlotinib Supplementary Table 2 Change of tumor biomarkers in tumor biopsies following treatment with lumretuzumab and cetuximab or erlotinib</p>
Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer. We believe that the strategy delineated here provides a path forward to avoid the time- and cost-consuming step of having to develop immunological reagents against unproven targets. We expect that mass spectrometry-based platforms may become part of a rational process to rapidly test and qualify large number of candidate biomarkers to identify the few that stand a chance for further development and validation.
Abstract Glofitamab, a novel CD20xCD3, T-cell–engaging bispecific antibody, exhibited single-agent activity in Study NP30179, a first-in-human, phase 1 trial in relapsed/refractory B-cell non-Hodgkin lymphoma. Preclinical studies showed that glofitamab leads to T-cell activation, proliferation, and tumor cell killing upon binding to CD20 on malignant cells. Here, we provide evidence of glofitamab’s clinical activity, including pharmacodynamic profile, mode of action, and factors associated with clinical response, by evaluating biomarkers in patient samples from the dose-escalation part of this trial. Patients enrolled in Study NP30179 received single-dose obinutuzumab pretreatment (1000 mg) 7 days before IV glofitamab (5 µg-25 mg). Glofitamab treatment lasted ≤12 cycles once every 2 or 3 weeks. Blood samples were collected at predefined time points per the clinical protocol; T-cell populations were evaluated centrally by flow cytometry, and cytokine profiles were analyzed. Immunohistochemical and genomic biomarker analyses were performed on tumor biopsy samples. Pharmacodynamic modulation was observed with glofitamab treatment, including dose-dependent induction of cytokines, and T-cell margination, proliferation, and activation in peripheral blood. Gene expression analysis of pretreatment tumor biopsy samples indicated that tumor cell intrinsic factors such as TP53 signaling are associated with resistance to glofitamab, but they may also be interlinked with a diminished effector T-cell profile in resistant tumors and thus represent a poor prognostic factor per se. This integrative biomarker data analysis provides clinical evidence regarding glofitamab’s mode of action, supports optimal biological dose selection, and will further guide clinical development. This trial was registered at www.clinicaltrials.gov as #NCT03075696.
Abstract Introduction: FAP-DR5 (RO6874813) is a novel bispecific antibody that binds with high and low affinity to fibroblast activation protein (FAP) and death receptor 5 (DR5), respectively. FAP-driven binding of RO6874813 mediates the high levels of DR5 clustering that are required for triggering cell death. Here, we present ongoing phase 1 data in patients with advanced solid tumors who were treated with escalating doses of single agent RO6874813 and assessed for tolerability. Methods: Study endpoints are safety and tolerability (primary) and antitumor activity (secondary). The study uses a continuous reassessment method (CRM) design for dose escalation. Patients received drug (IV ≤ 90 min) weekly (QW) or every other week (Q2W), starting with a run-in dose on Cycle 0/Day 1 (C0/D1) of 0.5 mg/kg for all cohorts to characterize linear and nonlinear pharmacokinetics (PK) . Doses administered at C1/D1 ranged from 1.0 to 45 mg/kg (3 or more patients per cohort). Dosing continued until progression of disease (PD) or toxicity occurred. Plasma biomarkers (BM) of DR5 binding (TRAIL, the DR5 ligand) and apoptosis (ccCK18) were measured at multiple timepoints. Archival or fresh tumor samples collected prior to RO6874813 treatment were analyzed for target expression by IHC and mRNA (qRT-PCR), cellular infiltrates, and apoptosis. Results: As of 26 April 2017, 32 patients have been treated with RO6874813. Patients had a median of 3.5 prior regimens (range 1-11) and received a median of 4 (range 2-21) doses of RO6874813. One patient (NSCLC) remains on treatment; 31 discontinued treatment (30 for PD; 1 for subject decision). A maximum tolerated dose has not been reached. The most common treatment-related adverse events (TR-AEs) were: fatigue (21.9%); nausea (15.6%), and infusion-related reactions (9.4%). Grade (Gr) ≥3 TR-AEs occurred in 2 patients (6.25%): anemia and asthenia (both Gr 3, occurring in 1 patient each). No Gr 4/5 TR-AEs and no protocol-defined DLTs were reported. No AE led to permanent study drug withdrawal; 5 patients died from PD, one within 30 days of their last dose. Thirty-one patients were evaluated for antitumor activity: using RECIST criteria, 1 PR (NSCLC; time on treatment = 324 days, ongoing) and 6 stable diseases (SD; median duration 42 days) were observed. 28 patients were evaluated by PET, with 2 (7%) FDG partial metabolic responses (EORTC criteria) seen. No difference was found between QW (used with select doses) and Q2W schedules for safety, antitumor activity, and PK/PD parameters. RO6874813 serum concentrations increased linearly with dose and revealed saturation of TMDD at ≥ 5 mg/kg (Q2W). For the single patient with a PR (30 mg/kg; Q2W), the Cmax at C1 exceeded that of other patients and showed accumulation over time, despite two dose interruptions for Gr 2 neutropenia. Blood BM analyses revealed a significant upregulation of TRAIL and ccCK18 after dosing in this and other patients, suggesting apoptotic activity. FAP and DR5 were expressed in tumor tissue of all patients. Conclusions: RO6874813 demonstrated a favorable safety profile in patients with multiple solid tumor types, and dose escalation and regimen optimization continue. Preliminary antitumor activity was observed in a patient with heavily pretreated NSCLC. Analyses required to support the hypothesis that FAP-binding mediates sufficiently high levels of DR5 clustering for apoptosis induction are ongoing. Citation Format: Johanna Bendell, Jean-Yves Blay, Philippe Cassier, Todd Bauer, Catherine Terret, Claudia Mueller, Anthony Morel, Evelyne Chesne, Zhi-xin Xu, Jean Tessier, Maurizio Ceppi, Ian James, Sabine Wilson, Elizabeth Quackenbush, Maria Ochoa de Olza, Josep Tabernero, Maria De Miguel, Emiliano Calvo. Phase 1 trial of RO6874813, a novel bispecific FAP-DR5 antibody, in patients with solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A092.
<p>Supplementary Figure 1 Quantile plots of baseline membrane HER3 IRS score (A) and HER3 mRNA log expression (B) in baseline FFPE tumor biopsy samples from patients with known adeno- and squamous cell carcinoma histology Supplementary Figure 2 Quantile plots of baseline membrane HER3 immuno-reactive scores in fresh FFPE tumor biopsies from NSCLC patients with non-squamous or squamous histology Supplementary Figure 3 Quantile plots of baseline HRG mRNA log expression determined by qRT-PCR in FFPE tumor biopsies of adeno- and squamous cell carcinoma histology (data obtained using the HRG research assay) Supplementary Figure 4 Baseline HRG mRNA log expression in FFPE tumor biopsies, determined by qRT-PCR, in non-sqNSCLC and sqNSCLC (data obtained using the HRG prototype diagnostic assay). Dotted line indicates prior determined median HRG expression in sqNSCLC. Supplementary Figure 5 Best percentage change from baseline in sum of longest diameters of target lesions for patients in the dose escalation and extension phase of the cetuximab part (n = 38). Red bars indicate PD patients, blue bars SD patients, light green bars PR patients and dark green bars CR patients. Eleven patients were without target lesion assessment post baseline. Tumor type and lumretuzumab dose are indicated. * indicates patients who received previous EGFR-targeting therapy. a indicates a patient who received prior trastuzumab therapy. Supplementary Figure 6 Best percentage change from baseline in sum of longest diameters of target lesions for patients in the dose escalation phase and the two NRG1 fusion gene patients of the extension phase of the erlotinib part (n = 31). Red bars indicate PD patients, blue bars SD patients and green bars PR patients. Eight patients were without target lesion assessment post baseline. Tumor type and lumretuzumab dose are indicated. * indicates patients who received previous EGFR-targeting therapy. a indicates two patients with NRG1 gene fusion.</p>
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