Abstract 1365: INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers
Authors: Bruni Sofia , Mercogliano Maria F. , Schillaci Roxana 
Published: March 2024

Abstract
Trastuzumab resistance is an important clinical issue in HER2+ breast cancer (BC), and few actionable targets are available. We demonstrated that soluble TNF (sTNF) upregulates mucin 4 (MUC4) expression, which shields trastuzumab epitope on HER2 hindering its therapeutic effect, and that sTNF blockade with INB03 decreases MUC4 expression which, together with trastuzumab, triggers an effective antitumor immune response based on antitumoral macrophage-NK cell collaboration. Because immune checkpoint molecules (ICP) and T cell exhaustion foster tumor immune escape, we addressed whether INB03 could modulate macrophage polarization, T cell exhaustion, and ICP expression in both populations to boost an antitumor immune response. For in vitro assays, human monocytes and T cells were isolated from buffy coats and enriched with specific RosetteSep. Macrophages (Mϕ) were differentiated for 6 days with M-CSF (10 ng/ml) to M0, and polarized for 48h to the M1-like subtype with LPS (50 ng/ml)+IFNγ (20 ng/ml), or to the M2-like subtype with IL-4 (20 ng/ml)+IL-10 (20 ng/ml). Then, INB03 was added (10 μg/ml) for 96h with corresponding cytokines to already-polarized Mϕ to test its ability to revert polarization. Also, Mϕ antibody-dependent cellular phagocytosis (ADCP) against human HER2+ breast cancer cell line JIMT-1 was assessed. T cells were activated with CD3/CD28 beads and cultured for 7 or 14 days. INB03 was added at those times for 48h to test its ability to modulate ICP and exhaustion markers. For in vivo assays, tumor-free mice or bearing the HER2+ C4HD tumor were treated with vehicle or INB03 (10 mg/kg, i.p.) for 21 days. Polarization, exhaustion and ICP markers were studied in Mϕ and T cells from spleen and tumor. Adding INB03 to already-polarized human Mϕ enhanced polarization towards M1-like (CD86+CD206−). Polarized Mϕ exposed to INB03 showed decreased PD1 and PDL-1 expression. Also, the expression of ADCP-inhibitory markers B7H4 and SIRPα was diminished when INB03 was present. These Mϕ exhibited increased ADCP against JIMT-1 cells treated with INB03, which showed downregulation of the inhibitory signal CD47. Murine Mϕ from tumor-bearing mice treated with INB03 showed increased polarization to the M1-like phenotype. Human CD8+ T cells showed decreased expression of PD1, PD-L1, LAG3, TIM3 and TIGIT. However, only TIM3 and PD1 were downregulated in murine CD8+ T cells of tumor-bearing mice treated with INB03. In all, sTNF blockade skews Mϕ to the M1-like phenotype and reprograms already-polarized pro-tumoral M2-like Mϕ to antitumoral ones. INB03 can tailor the tumor microenvironment by promoting Mϕ ADCP against tumor cells and by acting as an ICP inhibitor for CD8+ T cells, possibly relieving their exhaustion. HER2+MUC4+ BC patients could benefit from the administration of INB03 to boost targeted therapy efficacy and overcome tumor-induced macrophage and T-cell immune escape.
Citation Format: Sofia Bruni, Maria F. Mercogliano, Roxana Schillaci. INB03: a new immune checkpoint inhibitor that reprograms macrophage polarization, boosts ADCP and reverts T-cell exhaustion markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1365.