David Yoonsuk Oh

Purpose: Objective responses to peptide receptor radionuclide therapy (PRRT) for pretreated patients with well-differentiated neuroendocrine tumors (WD-NET) and a Ki-67 index >10% may not be durable. The response rate to single-agent immune checkpoint inhibitors (CPI) for patients with WD-NET is low. Targeted radiation using PRRT may potentiate the antitumor immune response. This study evaluated the safety and efficacy of the combination of PRRT and the PD-1 inhibitor pembrolizumab in high-risk WD-NET. Patients and Methods: In a single-arm prospective pilot study, adult patients with World Health Organization grade 2 or 3 well-differentiated (Ki-67 index >10%), somatostatin receptor-avid metastatic neuroendocrine tumors of any primary site received concurrent pembrolizumab and 177Lu-DOTATATE PRRT. The primary endpoint was the objective response rate (ORR) by RECIST v1.1. Secondary endpoints were progression-free survival (PFS), overall survival (OS), and safety. Results: A total of 26 patients were enrolled, including 20 patients with grade 3 neuroendocrine tumors. The median PFS was 13.3 months (range, 2.1–33.4 months). The ORR was 35%. The median OS was 24.1 months (range, 4.4–52.3 months). Severe adverse events (AE) occurred in 14 (54%) patients. The most common immune-related AE were grade 1 or 2 hepatitis (n = 7), grade 2 hypothyroidism (n = 5), and diabetes mellitus (n = 4). Clinical responses were associated with higher baseline frequencies of proliferating CD8+ T cells and lower frequencies of CD14+ myeloid-derived suppressor cells in the peripheral blood. Conclusions: The added benefit of CPI to PRRT for patients with high-risk WD-NET remains unclear. The severe AE rate was higher than expected for PRRT alone.

Figure S1, S2, S3, S4, S5, S6, S7, S8, S9, Table S1

Purpose:Chimeric antigen receptor (CAR) T-cell therapies have shown potential in solid tumors. A higher proportion of stem cell–like memory T cells (TSCM) in CAR T-cell products could enhance engraftment, persistence, and prolong immune activity. This phase I trial (NCT04249947) evaluated the safety and efficacy of P-PSMA-101, an autologous TSCM-rich, bone-tropic CAR T-cell therapy targeting prostate-specific membrane antigen (PSMA), in patients with metastatic castrate-resistant prostate carcinoma (mCRPC). Secondary endpoints included objective response rate, prostate-specific antigen (PSA) response, and radiographic progression-free survival. Patients and Methods:The final P-PSMA-101 product was produced from leukapheresis using the piggyBac DNA transposon–based platform, which integrates a multicistronic transgene encoding an inducible caspase 9 (iCasp9) safety switch in addition to the CAR, generating TSCM-rich CAR T cells. Results:Among 33 treated patients, 18% (n = 6) had dose-limiting toxicities. Cytokine release syndrome (CRS) occurred in 61% (n = 20), with grade ≥3 CRS seen in 9% (n = 3). Activation of the iCasp9-based safety switch was required in 24% (n = 8) of cases, including one toxicity that was ultimately fatal and successful resolution of symptoms in the other seven. P-PSMA-101 demonstrated antitumor activity, with 21% (n = 7) of patients achieving a ≥50% PSA decline (PSA50 response). Among 13 RECIST-evaluable patients, one partial response was observed. Stable disease was observed in 61% (n = 20) of patients, with 21% (n = 7) maintaining disease stability for ≥3 months. Two patients experienced sustained remissions exceeding 12 months, characterized by PSA declines of more than 90%, corroborated by pharmacokinetic, biomarker, and PSMA-PET imaging data. Conclusions:Robust expansion of P-PSMA-101 CAR T cells resulted in toxicity but also durable responses in patients with mCRPC. Future trials of CAR T therapy may be informed by the results of this nonviral engineering, TSCM cell–enriched approach.See related commentary by Lee et al., p. 3417

Imaging-based single-cell spatial transcriptomics (iSCST) on formalin-fixed, paraffin-embedded (FFPE) tissue enables comprehensive analysis of archived specimens while preserving spatial context, critical to an understanding of ulcerative colitis (UC) pathology. Here, we deployed a robust framework for applying iSCST to clinical FFPE mucosal biopsies from patients with UC, immune checkpoint inhibitor-induced (ICI) colitis and healthy controls. iSCST using custom Xenium gene panels enabled precise detection of diverse cell subsets and disease-specific genes. We mapped transcriptionally distinct fibroblast subsets within mucosal niches, including inflammation-associated fibroblasts (IAFs), and identified colitis-specific neighborhoods formed by IAFs, monocytes, and neutrophils. Transcriptional signatures and spatial neighborhoods uncovered through iSCST were associated with vedolizumab (VDZ) response, with non-responders exhibiting either an innate IAF-monocyte-neutrophil signature or adaptive gut-associated lymphoid tissue (GALT) signature, while responders showed enrichment of an epithelial cellular neighborhood. These signatures were validated in an internal and an external dataset, supporting the existence of two distinct archetypes of treatment resistance to VDZ in UC. This iSCST framework provides a powerful approach for analyzing FFPE tissues, offering insights into colitis-associated cellular networks and identifying biomarkers to enhance patient risk stratification in routine clinical workflows.

Purpose: Chimeric antigen receptor (CAR) T-cell therapies have shown potential in solid tumors. A higher proportion of stem cell–like memory T cells (TSCM) in CAR T-cell products could enhance engraftment, persistence, and prolong immune activity. This phase I trial (NCT04249947) evaluated the safety and efficacy of P-PSMA-101, an autologous TSCM-rich, bone-tropic CAR T-cell therapy targeting prostate-specific membrane antigen (PSMA), in patients with metastatic castrate-resistant prostate carcinoma (mCRPC). Secondary endpoints included objective response rate, prostate-specific antigen (PSA) response, and radiographic progression-free survival. Patients and Methods: The final P-PSMA-101 product was produced from leukapheresis using the piggyBac DNA transposon–based platform, which integrates a multicistronic transgene encoding an inducible caspase 9 (iCasp9) safety switch in addition to the CAR, generating TSCM-rich CAR T cells. Results: Among 33 treated patients, 18% (n = 6) had dose-limiting toxicities. Cytokine release syndrome (CRS) occurred in 61% (n = 20), with grade ≥3 CRS seen in 9% (n = 3). Activation of the iCasp9-based safety switch was required in 24% (n = 8) of cases, including one toxicity that was ultimately fatal and successful resolution of symptoms in the other seven. P-PSMA-101 demonstrated antitumor activity, with 21% (n = 7) of patients achieving a ≥50% PSA decline (PSA50 response). Among 13 RECIST-evaluable patients, one partial response was observed. Stable disease was observed in 61% (n = 20) of patients, with 21% (n = 7) maintaining disease stability for ≥3 months. Two patients experienced sustained remissions exceeding 12 months, characterized by PSA declines of more than 90%, corroborated by pharmacokinetic, biomarker, and PSMA-PET imaging data. Conclusions: Robust expansion of P-PSMA-101 CAR T cells resulted in toxicity but also durable responses in patients with mCRPC. Future trials of CAR T therapy may be informed by the results of this nonviral engineering, TSCM cell–enriched approach.

Purpose: We report results from two clinical trials of the cyclic dinucleotide stimulator of IFN genes (STING) agonist ulevostinag. Patients and Methods: In a phase I study (NCT03010176) with an accelerated titration design/modified toxicity probability interval method, participants with advanced/metastatic solid tumors or lymphomas received intratumoral ulevostinag (±intravenous pembrolizumab). In an expansion phase, participants with head and neck squamous cell carcinoma (HNSCC) or triple-negative breast cancer received the combination. Primary objectives were safety/tolerability and identifying the recommended phase II dose; biomarkers were exploratory. In a randomized phase II study (NCT04220866), participants with untreated metastatic or unresectable, recurrent HNSCC received intravenous pembrolizumab (±ulevostinag 540 µg). The primary objective was antitumor activity. Pembrolizumab 200 mg was administered every 3 weeks in both studies. Results: In the phase I study (NCT03010176; N = 156), the most common adverse event was pyrexia (70%). Plasma ulevostinag concentrations increased dose-dependently. Circulating levels of C-X-C motif chemokine 10, IFNγ, and IL-6 showed elevation at 2 to 4 hours, peak at 6 to 8 hours, and plateau/partial resolution at 24 hours but, beyond the 540 µg dose, did not show a clear dose–effect relationship. Ten participants experienced dose-limiting toxicities; the recommended phase II dose for intratumoral ulevostinag was 540 µg. In the phase II study (NCT04220866), 4 of 8 participants treated with combination therapy and 1 of 10 treated with pembrolizumab monotherapy had a complete or partial response. The most common adverse event was pyrexia (n = 5). Conclusions: Intratumoral ulevostinag (±pembrolizumab) had manageable toxicity, dose-dependent pharmacokinetics, and evidence of STING activation and target engagement. Combination therapy showed antitumor activity in participants with untreated metastatic or unresectable, recurrent HNSCC.

While cytotoxic CD4 ⁺ tumor-infiltrating lymphocytes have anticancer activity in patients, whether these can be noninvasively monitored and how these are regulated remains obscure. By matching single cells with T cell receptors (TCRs) in tumor and blood of patients with bladder cancer, we identified distinct pools of tumor-matching cytotoxic CD4 ⁺ T cells in the periphery directly reflecting the predominant antigenic specificities of intratumoral CD4 ⁺ tumor-infiltrating lymphocytes. On one hand, the granzyme B–expressing (GZMB-expressing) cytotoxic CD4 ⁺ subset proliferated in blood in response to PD-1 blockade but was separately regulated by the killer cell lectin-like receptor G1 (KLRG1), which inhibited their killing by interacting with E-cadherin. Conversely, a clonally related, GZMK-expressing circulating CD4 ⁺ population demonstrated basal proliferation and a memory phenotype that may result from activation of GZMB ⁺ cells, but was not directly mobilized by PD-1 blockade. As KLRG1 marked the majority of circulating tumor-TCR-matched cytotoxic CD4 ⁺ T cells, this work nominates KLRG1 as a means to isolate them from blood and provide a window into intratumoral CD4 ⁺ recognition, as well as a putative regulatory receptor to mobilize the cytolytic GZMB ⁺ subset for therapeutic benefit. Our findings also underscore ontogenic relationships of GZMB- and GZMK-expressing populations and the distinct cues that regulate their activity.

Background & Aims: Imaging-based, single-cell spatial transcriptomics (iSCST) using formalin-fixed, paraffin-embedded (FFPE) tissue could transform translational research by retaining all tissue cell subsets and spatial locations while enabling the analysis of archived specimens. We aimed to develop a robust framework for applying iSCST to archived clinical FFPE mucosal biopsies from patients with inflammatory bowel disease (IBD). Methods: We performed a comprehensive benchmarking comparison of three iSCST platforms capable of analyzing FFPE specimens. We analyzed FFPE mucosal biopsies (n=57) up to 5 years old from non-IBD controls (HC; n=9) and patients with ulcerative colitis (UC;n=11). After platform-specific cell segmentation, we applied a uniform data processing pipeline to all datasets, including transcript detection, cell annotation, differential gene expression, and neighborhood enrichment. Transcriptomic signatures identified with iSCST were validated using external, publicly available bulk transcriptomic datasets. Results: A custom 290-plex Xenium gene panel exhibited the highest sensitivity and specificity for transcript detection, enabling precise identification and quantification of diverse cell subsets and differentially expressed genes across cell types and disease states. We mapped transcriptionally distinct fibroblast subsets to discrete spatial locations and identified inflammation-associated fibroblasts (IAFs) and monocytes as a colitis-associated cellular neighborhood. We also identified signatures associated with Vedolizumab (VDZ) responsiveness. VDZ non-responders were characterized by an IAF-monocyte transcriptional signature, while responders exhibited enrichment of epithelial gene sets. Conclusions: Our optimized iSCST framework for archived FFPE biopsies provides unique advantages for assessing the role of colitis-associated cellular networks in routinely collected clinical samples. FFPE-based biomarkers could integrate with existing clinical workflows and potentially aid in risk-stratifying patients.

Identifying pan-tumor biomarkers that predict responses to immune checkpoint inhibitors (ICI) is critically needed. In the AMADEUS clinical trial (NCT03651271), patients with various advanced solid tumors were assessed for changes in intratumoral CD8 percentages and their response to ICI. Patients were grouped based on tumoral CD8 levels: those with CD8 <15% (CD8-low) received nivolumab (anti-PD-1) plus ipilimumab (anti-CTLA4) and those with CD8 ≥15% (CD8-high) received nivolumab monotherapy. 79 patients (72 CD8-low and 7 CD8-high) were treated. The disease control rate was 25.0% (18/72; 95% CI: 15.8–35.2) in CD8-low and 14.3% (1/7; 95% CI: 1.1–43.8) in CD8-high. Tumors from 35.9% (14/39; 95% CI: 21.8–51.4) of patients converted from CD8 <15% pretreatment to ≥15% after treatment. Multiomic analyses showed that CD8-low responders had an inflammatory tumor microenvironment pretreatment, enhanced by an influx of CD8 T cells, CD4 T cells, B cells, and macrophages upon treatment. These findings reveal crucial pan-cancer immunological features for ICI response in patients with metastatic disease.

Background: Colitis caused by checkpoint inhibitors (CPI) is frequent and is treated with empiric steroids, but CPI colitis mechanisms in steroid-experienced or refractory disease are unclear.Methods: Using colon biopsies and blood from predominantly steroid-experienced CPI colitis patients, we performed multiplexed single-cell transcriptomics and proteomics to nominate contributing populations.Results: CPI colitis biopsies showed enrichment of CD4⁺resident memory (RM) T cells in addition to CD8⁺RM and cytotoxic CD8⁺T cells. Matching T cell receptor (TCR) clonotypes suggested that both RMs are progenitors that yield cytotoxic effectors. Activated, CD38⁺HLA-DR⁺CD4⁺RM and cytotoxic CD8⁺T cells were enriched in steroid-experienced and a validation data set of steroid-naïve CPI colitis, underscoring their pathogenic potential across steroid exposure. Distinct from ulcerative colitis, CPI colitis exhibited perturbed stromal metabolism (NAD⁺, tryptophan) impacting epithelial survival and inflammation. Endothelial cells in CPI colitis after anti-TNF and anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) upregulated the integrin α4β7 ligand molecular vascular addressin cell adhesion molecule 1 (MAdCAM-1), which may preferentially respond to vedolizumab (anti-α4β7).Conclusions: These findings nominate CD4⁺RM and MAdCAM-1⁺endothelial cells for targeting in specific subsets of CPI colitis patients.