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Background: Signal Regulatory Protein α [SIRPα] is an inhibitory membrane receptor expressed by myeloid cells (macrophages and myeloid-derived suppressor cells, MDSCs) and specifically binds to CD47. BI765063 is a selective anti-SIRPα monoclonal antibody acting as a checkpoint inhibitor of SIRPα/CD47 axis, promoting anti-tumor immunity through increase of dendritic cells antigen presentation, enabling MDSC differentiation, potentiating macrophage phagocytic/inflammatory properties and reinstating myeloid cell chemokine secretion and human T cell migration1. The escalation phase I study in advanced solid tumor patients (pts) showed preliminary efficacy as monotherapy (MONO), and in combination with PD-1 inhibitor ezabenlimab (COMBO). Our goal was to investigate BI765063 predictive response markers. Methods: A total of 68 patients (50 in MONO arm, 18 in COMBO arm) have been enrolled. Receptor occupancy (RO) was determined on peripheral CD14+ monocytes. Tumor biopsies were collected before treatment from 44 pts (62%) representative of overall population. Tumor microenvironment (TME) was analysed using a Brightplex® IHC panel comprised of CD68+ macrophages, CD11b+myeloid cells, SIRPα, and CD47. NanoString tumor profiling used PanCancer IO360 panel. Results: One partial response (PR) in MONO (Hepatocellular carcinoma HCC), and 4 PRs in COMBO (2 endometrial cancer, 1 HCC, 1 iRecist PR in MSS CRC) were observed2. BI 765063 full RO saturation was achieved in V1/V1 pts treated with doses of 6 mg/kg and higher, while V1/V2 patients showed a more heterogeneous RO ranging between 40-80%, reaching an apparent saturation at doses of 12 mg/kg and higher. Comparison of pts by best response showed that baseline tumor SIRPα+ expression in CD68+ macrophages and in CD11b+ myeloid cells were higher in PR versus PD pts. High percentage of CD11b+SIRPα+ myeloid cells at baseline significantly correlated with better OS (p=0.023), while CD68+SIRPα+ macrophages high expression in TME showed a trend for a better OS but did not reach statistical significance. The CD47 tumor expression did not correlate with the OS. Responder signature based on the top deregulated genes in responders versus non-responders at baseline was devised to sort relevant TCGA cohorts and showed strong positive correlation with TME MDSC infiltration (p≤0.0001). Conclusions: High levels of CD11b+SIRPα+ myeloid cells in TME at baseline, but not CD47 tumor expression, correlates with longer survival while MDSC signature in TME at baseline correlates with clinical response. Thus, suggesting that MDSCs expressing SIRPα in TME could represent a predictive efficacy biomarker. References: 1. Gauttier V. et al., 2020. J. Clin. Invest. 130: 6109; 2. Kotecki N. et al., 2021. ESMO meeting, abstract #983P Citation Format: Stéphane Champiat, Philippe A. Cassier, Nuria Kotecki, Carlos Gomez-Roca, Iphigenie Korakis, Ouali Kaissa, Antoine Italiano, Mabrouk M. Elgadi, Thomas Vandewalle, Isabelle Girault, Nina Salabert-Le Guen, Donogh O’Brien, Nicolas Poirier, Bérangère Vasseur, Dominique Costantini, Claudia Fromond, Françoise Bono, Jean-Pierre Delord. Predictive response biomarkers from Phase I clinical trial of a SIRPalpha inhibitor BI765063, stand-alone and in combination with ezabenlimab, a PD1 inhibitor, in patients with advanced solid tumors [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 2129.

Background: BI 765063 (OSE-172) is a humanised IgG4 monoclonal antibody which binds selectively to the V1 allele of Signal Regulatory Protein α [SIRPα] blocking the SIRPα/CD47 “don't eat me” pathway. Preclinical studies showed that SIRPα blockage led to macrophage and T-cell recruitment into tumor xenografts, and induced upregulation of chemokines, cytokines and adaptive immune function genes in human tumor explants (Gauttier et al., 2020). The goal of the biomarker analyses was to characterize the BI 765063 impact on peripheral blood immune cells (PBMCs) and the tumor microenvironment (TME). Methods: Fifty patients (26 V1/V1, 24 V1/V2) received BI 765063 IV from 0.02 mg/kg to 36 mg/kg every 3 weeks. Paired tumor biopsies were collected before and 2 weeks after first BI 765063 infusion. PBMCs were collected before, then 4 h, 1, 14, and 21 days after first infusion. BI 765063 receptor occupancy (RO) was determined on peripheral CD14+ monocytes. Immunophenotyping of PBMCs was performed by flow-cytometry. TME was analysed with a Brightplex® IHC panel including CD8+ T-cells, CD68+ macrophages, SIRPα, CD47, and PD-L1. Tumor gene expression profiling was performed using the Pan Cancer Immune gene set. Results: BI 765063 full RO saturation was achieved at trough levels (C2D1, pre-dose) in V1/V1 patients treated with doses of 6 mg/kg and higher, while V1/V2 patients showed a more heterogeneous RO ranging from 40-80%, reaching an apparent saturation at ≥ 12 mg/kg. An increase of activated CD80+/CD14+ and CD40+/CD14+ monocytes in PBMCs was observed at 24 h post-treatment in both, V1/V1 and V1/V2 patients. In paired tumor biopsies, IFNγ, MHCII antigen presentation gene pathways, and CCL7 transcripts appeared to be upregulated at C1D15 in patients with a systemic exposure of ≥ 100 µg/ml. One patient with hepatocellular carcinoma (HCC) and liver and lung metastases treated with BI 765063 monotherapy at 24 mg/kg achieved partial response (Champiat et al., ASCO, 2021). Baseline tumor biopsy of that patient showed that 66% of HCC tumor cells were CD47+ and 87% of CD68+ macrophages were SIRPα+. Furthermore, high levels of CD8+ T-cells were observed at baseline. At C1D15 increased CD68+ macrophage infiltration, sustained CD8 T-cell tumor accumulation and higher PD-L1 CPS (48% at baseline vs 75% at C1D15) were observed. Analysis of paired tumor biopsies in other patients showed that often, increased levels of tumor CD68+ macrophages were accompanied by CD8+ T-cell infiltration. Conclusion: This early biomarker analysis in patients with a wide range of solid tumors and treated with the first-in-class SIRPa inhibitor BI 765063 show encouraging signs of potentially mode-of-action related changes, both in peripheral blood and the TME. These early signals will be further evaluated in similar samples from the ongoing expansion cohorts in more homogeneous patient populations. Citation Format: Stephane Champiat, Philippe A. Cassier, Nuria Kotecki, Carlos Gomez-Roca, Aurélien Marabelle, Armelle Vinceneux, Christiane Jungels, Mabrouk Elgadi, Ralph Graeser, Thomas Vandewalle, Isabelle Girault, Nina Salabert-Le Guen, Nicolas Poirier, Bérangère Vasseur, Dominique Costantini, Claudia Fromond, Jean-Pierre Delord. Biomarker analyses from the Phase I clinical trial of the first-in-class SIRPa immune checkpoint inhibitor BI765063 in patients with advanced solid tumors [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 1993.

BackgroundImmune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Besides, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent by which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods.MethodsIn this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC.ResultsThough partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa).ConclusionsIn conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.

Functional immune responses are increasingly important for clinical studies, providing in depth biomarker information to assess immunotherapy or vaccination. Incorporating functional immune assays into routine clinical practice has remained limited due to challenges in standardizing sample preparation. We recently described the use of a whole blood syringe-based system, TruCulture (R), which permits point-of-care standardized immune stimulation. Here, we report on a multi-center clinical study in seven FOCIS Centers of Excellence to directly compare TruCulture to conventional PBMC methods. Whole blood and PBMC5 from healthy donors were exposed to LPS, anti-CD3 anti-CD28 antibodies, or media alone. 55 protein analytes were analyzed centrally by Luminex multi-analyte profiling in a CLIA-certified laboratory. TruCulture responses showed greater reproducibility and improved the statistical power for monitoring differential immune response activation. The use of TruCulture addresses a major unmet need through a robust and flexible method for immunomonitoring that can be reproducibly applied in multi-center clinical studies. One sentence summary: A multi-center study revealed greater reproducibility from whole blood stimulation systems as compared to PBMC stimulation for studying induced immune responses. (C) 2017 Published by Elsevier Inc.

International audience

International audience; Objective: Dendritic cells (DCs) are critical effectors of innate and adaptive immunity playing crucial roles in autoimmune responses. We previously showed that blood DC numbers were reduced in autoimmune antineutrophil cytoplasmic autoantibody-associated vasculitis (AAV). Here, we assessed toll-like receptor (TLR) responsiveness of blood DCs from patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA).Methods: Blood samples from healthy controls (HCs), GPA, or MPA patients, without treatment, during acute phase (AP) or remission phase (RP) were analyzed. Cytokine production by DCs and T cells was assessed on whole blood by flow cytometry after TLRs or polyclonal stimulation, respectively.Results: We first showed that GPA and MPA are associated with a decreased blood DC number during AP. Conventional DCs (cDCs) from patients with GPA and MPA in AP exhibited a profound decrease of IL-12/IL-23p40 production after TLR3, 4, or 7/8 stimulation compared to patients in remission and HC, with a return to normal values in RP. TNFα secretion was also affected, with a decrease in cDCs from GPA patients in AP after TLR3 stimulation but an increase after TLR7/8 stimulation. By contrast, the responsiveness of plasmacytoid DCs to TLR7 and 9 was only marginally affected. Finally, we observed that IFNγ-producing CD4 + T cell frequency was significantly lower in AP-GPA patients than in HC.Conclusion: We describe, for the first time, a dysregulated response to TLRs of circulating DCs in AAV patients mostly affecting cDCs that exhibit an unexpected reduced inflammatory cytokine secretion possibly contributing to an altered Th cell response.