Brandie C. Taylor

Purpose:Immune checkpoint inhibitors (ICI) have increasing application in endometrial cancer, underscoring the need for robust biomarkers for patient selection. JAK1 pathogenic variants (PV) have previously been implicated in immune evasion. In this study, we identify biomarkers predictive of ICI response in endometrial cancer and the implications of JAK1 PVs in this context.Experimental Design:This is a translational study of tumors from 84 patients with endometrial cancer treated with ICIs. High-throughput proteomic-based profiling was used to quantify 193 phosphoprotein/protein expression levels, including key JAK/STAT signaling pathway components. Associations with clinical outcomes were assessed using multivariate regression analysis. The functional consequences of JAK1 PVs were explored through in vitro signaling assays and analyses of The Cancer Genome Atlas database.Results:MHC-I expression correlated with improved progression-free survival (P = 0.035), validated in orthogonal approaches. Notably, a subset of patients harboring JAK1 PVs demonstrated exceptional survival on ICIs. In The Cancer Genome Atlas cohort of microsatellite instability–high and DNA polymerase epsilon–mutated tumors, homozygous loss of JAK1 trended toward decreased survival, whereas heterozygous loss of JAK1 was associated with significantly improved survival (P = 0.026), suggesting partial retention of antigen presentation pathways. Among our ICI-treated microsatellite instability–high tumor samples, NK cell marker NCAM1 was associated with improved survival (P = 0.02).Conclusions:These data support MHC-I as a potential predictive biomarker for ICI response in endometrial cancer. Additionally, we show that partial retention of JAK1 signaling in JAK1 tumors with heterozygous loss is associated with improved survival, potentially attributable to enhanced NK cell activity in tumors with low MHC-I expression.

Background: Immune checkpoint inhibitors (ICI) have improved survival in various cancers, but their success in breast cancer, specifically triple-negative breast cancer, remains limited, benefiting less than 10% of patients. Methods: We modeled ICI response in vivo to unravel the mechanisms underlying immunotherapy efficacy, identify mechanisms of resistance in non-responsive tumors, and ascertain the therapeutic benefits of different chemotherapeutic combinations with ICI in breast cancer. We investigated the impact of ICI as monotherapy and in combination with other therapeutics in mouse models of mammary cancer, which we found robustly suppressed primary tumor growth and extended survival. Results: Interestingly, even within a single model, responses to ICI were highly variable. Resistance was not reliably retained by transplantation into syngeneic hosts, suggesting a role for systemic host immunity rather than tumor-autonomous mechanisms. Transcriptomic analysis of the primary tumor landscape by fine-needle aspiration revealed that upregulated cytotoxic T-cell response and inflammatory interferon signaling (both at baseline and post anti-programmed death-ligand 1 administration) corresponded to favorable response to ICI. Longitudinal analysis of the peripheral blood uncovered enhanced myeloid cell recruitment in resistant mice, prior to therapy initiation. Similar effects were observed through longitudinal assessment of peripheral blood in patients with ICI-treated human breast cancer. Blocking myeloid cell recruitment with navaraxin (CXCR1/2 inhibitor) improved ICI responses, further suppressing tumor growth and improving survival. Conclusions: These findings provide insight into resistance mechanisms and suggest the potential for minimally invasive strategies (sampling of systemic immune cells from peripheral blood) to identify patients likely to respond to ICI. This approach may help inform de-escalation strategies to mitigate therapeutic toxicities and limit unnecessary treatments.

Purpose: Immune checkpoint inhibitors (ICI) have increasing application in endometrial cancer, underscoring the need for robust biomarkers for patient selection. JAK1 pathogenic variants (PV) have previously been implicated in immune evasion. In this study, we identify biomarkers predictive of ICI response in endometrial cancer and the implications of JAK1 PVs in this context. Experimental Design: This is a translational study of tumors from 84 patients with endometrial cancer treated with ICIs. High-throughput proteomic-based profiling was used to quantify 193 phosphoprotein/protein expression levels, including key JAK/STAT signaling pathway components. Associations with clinical outcomes were assessed using multivariate regression analysis. The functional consequences of JAK1 PVs were explored through in vitro signaling assays and analyses of The Cancer Genome Atlas database. Results: MHC-I expression correlated with improved progression-free survival (P = 0.035), validated in orthogonal approaches. Notably, a subset of patients harboring JAK1 PVs demonstrated exceptional survival on ICIs. In The Cancer Genome Atlas cohort of microsatellite instability–high and DNA polymerase epsilon–mutated tumors, homozygous loss of JAK1 trended toward decreased survival, whereas heterozygous loss of JAK1 was associated with significantly improved survival (P = 0.026), suggesting partial retention of antigen presentation pathways. Among our ICI-treated microsatellite instability–high tumor samples, NK cell marker NCAM1 was associated with improved survival (P = 0.02). Conclusions: These data support MHC-I as a potential predictive biomarker for ICI response in endometrial cancer. Additionally, we show that partial retention of JAK1 signaling in JAK1 tumors with heterozygous loss is associated with improved survival, potentially attributable to enhanced NK cell activity in tumors with low MHC-I expression.

The enhancement of antitumour immunity via agonists of the stimulator of interferon genes (STING) pathway is limited by pharmacological barriers. Here we show that the covalent conjugation of a STING agonist to anti-albumin nanobodies via site-selective bioconjugation chemistries prolongs the circulation of the agonist in the blood and increases its accumulation in tumour tissue, stimulating innate immune programmes that increased the infiltration of activated natural killer cells and T cells, which potently inhibited the growth of mouse tumours. The technology is modular, as demonstrated by the recombinant integration of a second nanobody domain targeting programmed death-ligand 1 (PD-L1), which further increased the accumulation of the agonist in tumours while blocking immunosuppressive PD-1/PD-L1 interactions. The bivalent nanobody–STING agonist conjugate stimulated robust antigen-specific T-cell responses and long-lasting immunological memory and conferred enhanced therapeutic efficacy. It was also effective as a neoadjuvant treatment to adoptive T-cell therapy. As a modular approach, hitchhiking STING agonists on serum albumin may serve as a broadly applicable strategy for augmenting the potency of systemically administered cancer immunotherapies.

Stimulator of interferon genes (STING) is a promising target for potentiating antitumor immunity, but multiple pharmacological barriers limit the clinical utility, efficacy, and/or safety of STING agonists. Here we describe a modular platform for systemic administration of STING agonists based on nanobodies engineered for in situ hitchhiking of agonist cargo on serum albumin. Using site-selective bioconjugation chemistries to produce molecularly defined products, we found that covalent conjugation of a STING agonist to anti-albumin nanobodies improved pharmacokinetics and increased cargo accumulation in tumor tissue, stimulating innate immune programs that increased the infiltration of activated natural killer cells and T cells, which potently inhibited tumor growth in multiple mouse tumor models. We also demonstrated the programmability of the platform through the recombinant integration of a second nanobody domain that targeted programmed cell death ligand-1 (PD-L1), which further increased cargo delivery to tumor sites while also blocking immunosuppressive PD-1/PD-L1 interactions. This bivalent nanobody carrier for covalently conjugated STING agonists stimulated robust antigen-specific T cell responses and long-lasting immunological memory, conferred enhanced therapeutic efficacy, and was effective as a neoadjuvant treatment for improving responses to adoptive T cell transfer therapy. Albumin-hitchhiking nanobodies thus offer an enabling, multimodal, and programmable platform for systemic delivery of STING agonists with potential to augment responses to multiple immunotherapeutic modalities.

Anti-PD-L1 did not induce a proinflammatory immune response in B7-H4+ tumors. CD45+ cells were sorted from EMT6 tumors ± B7-H4 and subjected to NanoString gene expression analysis using the Mouse Pan-Cancer Immune Panel. A, Differentially expressed genes from CD45+ sorted cells from EMT6 control tumors when treated with anti-PD-L1 or isotype control and harvested 7 days after treatment. B, Changes in immune gene sets between isotype and anti-PD-L1–treated tumors. C, Differentially expressed genes from CD45+ sorted cells from EMT6-B7-H4+ tumors when treated with anti-PD-L1 or isotype control and harvested 7 days after treatment. D, Changes in immune gene sets between treatment groups of EMT6-B7-H4+ tumors. Data were analyzed by Wilcoxon rank-sum test. Genes with log2 fold change >0.5 or <−0.5 and P-value <0.01 were regarded as significant. n = 6 mice per group for all groups.

Supplemental Figure 7. B7-H4 and PD-L1 are not mutually exclusive on tumor cells. Data shown are reverse phase protein array expression data of B7-H4 and PD-L1 (SP142, 22C3, or Atezolizumab) from the I-SPY2 patient cohort. Samples were a mixture of TNBC and ER+ tumors and were treated with chemotherapy ± anti-PD-1. Depending on the antibody clone selected to detect PD-L1 expression, B7-H4 had no correlation, or a positive correlation to PD-L1 expression in these tumors. Data analyzed by spearman correlation and shown with linear regression best-fit line. n = 151 patients.

Anti-PD-L1 did not induce a proinflammatory immune response in B7-H4+ tumors. CD45+ cells were sorted from EMT6 tumors ± B7-H4 and subjected to NanoString gene expression analysis using the Mouse Pan-Cancer Immune Panel. A, Differentially expressed genes from CD45+ sorted cells from EMT6 control tumors when treated with anti-PD-L1 or isotype control and harvested 7 days after treatment. B, Changes in immune gene sets between isotype and anti-PD-L1–treated tumors. C, Differentially expressed genes from CD45+ sorted cells from EMT6-B7-H4+ tumors when treated with anti-PD-L1 or isotype control and harvested 7 days after treatment. D, Changes in immune gene sets between treatment groups of EMT6-B7-H4+ tumors. Data were analyzed by Wilcoxon rank-sum test. Genes with log2 fold change >0.5 or <−0.5 and P-value <0.01 were regarded as significant. n = 6 mice per group for all groups.

Supplemental Figure 4. B7-H4 expression did not change quantity of infiltrating tumor immune cells in vivo regardless of anti-PD-L1 treatment. Untreated and anti-PD-L1 treated EMT6 tumors ± B7-H4 were dissociated to single cell suspension and subjected to flow cytometry with a 14 (for myeloid cells) or 17 (for T cells) color panel on a CyTEK Aurora. n = 3/group for control and 8/group for treated samples. Data were analyzed by One-way ANOVA with Sidak’s post-hoc test for multiple comparisons between the EMT6 control anti-PD-L1 and EMT6 B7-H4+ anti-PD-L1 treatment groups. One-way ANOVA was not significant between groups. Data were analyzed in GraphPad Prism v10.