Dong, Haidong

Background Patients with metastatic melanoma rely on PD-(L)1 immunotherapy, but only one-third of patients experience treatment response and all initial responders eventually develop resistance. Tumour-derived extracellular vesicles expressing Programmed death ligand 1 (evPD-L1) and soluble Programmed death ligand 1 (sPD-L1) in peripheral blood of patients with melanoma limit PD-(L)1 immunotherapy and correlate with poor survival. Therapeutic plasma exchange (TPE) removes immunosuppressive evPD-L1 and sPD-L1. We hypothesise that TPE may rescue and restore antimelanoma immunity. Methods In this two-arm study, 60 patients with metastatic melanoma progressing on checkpoint inhibition will be accrued. All patients will undergo radiotherapy on days 1–5 (at least one measurable lesion will not be irradiated) and ongoing checkpoint inhibition on day 8 and every 2–3 weeks per standard of care. Patients with baseline sPD-L1 level of ≥1.7 ng/mL and adequate clinical capacity will be enrolled in the TPE intervention arm and will undergo TPE on days 5–7, in addition to standard of care radiotherapy and immunotherapy. Other patients will remain in the standard of care arm. The primary endpoint of the study is to evaluate safety. Secondary endpoints include kinetics of sPD-L1 and evPD-L1 and clinical response by RECIST (Response Evaluation Criteria in Solid Tumors) criteria. Study registered at ClinicalTrials.gov ( NCT04581382 ). Ethics and dissemination This trial has been approved by the Mayo Clinic Institutional Review Board. It will assess the safety and feasibility of TPE in improving outcomes for PD-(L)1 inhibitor immunotherapy in melanoma. Data will be maintained on a secure database with deidentified patient information. Data will be shared on publication in a peer-reviewed journal without the aid of professional writers. If successful, this trial will lay the ground for phase II studies that will include cancer treated with PD-(L)1 inhibitors which may benefit from TPE such as renal, bladder and lung cancers. Trial registration number NCT04581382 .

Abstract Purpose: This phase Ib/2 trial investigated pembrolizumab-containing trimodality therapy in patients with gastroesophageal junction (GEJ) adenocarcinoma. Patients and Methods: Patients with GEJ adenocarcinoma (cT1–3NanyM0) received neoadjuvant pembrolizumab-containing chemoradiation (CROSS regimen) followed by surgical resection and adjuvant pembrolizumab. The primary endpoints were tolerability in the first 16 patients and pathologic complete response [pCR (ypT0N0)]. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). An independent propensity-score-matched cohort (treated with CROSS without immunotherapy) was used for comparison. Exploratory analyses included immune biomarkers in the tumor microenvironment (TME) and plasma. Results: We enrolled 31 eligible patients, of whom 29 received all expected doses of neoadjuvant pembrolizumab and 28 underwent R0 resection. Safety endpoints were met. The primary efficacy endpoint was not met [7/31 (22.6%) achieved pCR]. Patients with high [i.e., combined positive score (CPS) ≥ 10] baseline expression of programmed death (PD)-L1 in the TME had a significantly higher pCR rate than those with low expression [50.0% (4/8) vs. 13.6% (3/22); P = 0.046]. Patients with high PD-L1 expression also experienced longer PFS and OS than propensity-score-matched patients. Among trial patients with PD-L1 CPS < 10, unprespecified analysis explored whether extracellular vesicles (EV) could identify further responders: an elevated plasma level of PD-L1–expressing EVs was significantly associated with higher pCR. Conclusions: Adding pembrolizumab to trimodality therapy showed acceptable tolerability but did not meet the pre-specified pCR endpoint. Exploratory analyses suggested that high PD-L1 expression in the TME and/or on EVs may identify patients most likely to achieve tumor response.

Abstract Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells that recognize specific sialic acid modifications on cell surface glycans. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered immune responses and increased tumor growth. In this study, we examined the role of ST8Sia6 on immune cells in regulating antitumor immunity. ST8Sia6 knockout mice had an enhanced immune response to tumors. The loss of ST8Sia6 promoted an enhanced intratumoral activation of macrophages and dendritic cells, including upregulation of CD40. Intratumoral regulatory T cells exhibited a more inflammatory phenotype in ST8Sia6 knockout mice. Using adoptive transfer studies, the change in regulatory T cell phenotype was not cell intrinsic and depended on the loss of ST8Sia6 expression in APCs. Thus, ST8Sia6 generates ligands for Siglecs that dampen antitumor immunity.

AbstractImmune checkpoint inhibitors (ICIs), as a novel class of anticancer therapy, can be more efficacious and less toxic than chemotherapy, but their clinical success is confined to certain tumor types. Elucidating their targets, mechanisms and scope of action, and potential synergism with chemotherapy and/or targeted therapies are critical to widen their clinical indications. Treatment response to an ICI targeting programmed death‐1 (anti‐PD‐1) is sought to be understood here by conducting a preplanned correlative analysis of a phase II clinical trial in patients with small bowel adenocarcinoma (SBA). The cytolytic capacity of circulating immune cells in cancer patients using a novel ex vivo cytotoxicity assay is evaluated, and the utility of circulating biomarkers is investigated to predict and monitor the treatment effect of anti‐PD‐1. Baseline expression of Bim and NKG7 and upregulation of CX3CR1 in circulating T cells are associated with the clinical benefit of anti‐PD‐1 in patients with SBA. Overall, these findings suggest that the frequency and cytolytic capacity of circulating, effector immune cells may differentiate clinical response to ICIs, providing a strong rationale to support immune monitoring using patient peripheral blood.

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr−/− mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr−/− mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. However, coculture of T cells with SB cells or SB cells deficient in Tr (SB-Tr−/−) did not result in a significant difference in T cell apoptosis or function, implying that TRAIL-TR is not a direct T cell checkpoint. Myeloid derived suppressor cells (MDSCs) were significantly decreased in Tr−/− tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail−/−) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. Coculture of SB cells with MDSCs from Tr−/− mice attenuated MDSC proliferation and immunosuppression compared to WT MDSCs. Moreover, treatment of MDSCs from Tr−/− mice with TRAIL recombinant protein resulted in a reduction in their proliferation and immunosuppressive function compare to MDSCs from WT mice implying that TRAIL-TR fosters MDSC growth and immunosuppressive function. In conclusion, we have demonstrated that Tr−/− mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr−/− mice bearing tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumor immune evasion via MDSCs. Herein, we suggest that TRAIL-TR appears to function as an indirect T cell checkpoint by augmenting the immunosuppressive effects of MDSCs. Citation Format: Emilien Loeuillard, Juan Wang, Jingchun Yang, Haidong Dong, Gregory Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) foster myeloid-derived suppressor cell-mediated tumor immune evasion in cholangiocarcinoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P072.

Abstract Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti–PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell–mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti–PD-1 or anti–PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen–specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell–intrinsic therapeutic target for enhancing cancer immunotherapy. See related article by Li et al., p. 154.

Abstract Background: Chimeric antigen receptor T cell therapy (CAR-T) is a recent therapeutic advance for the treatment of myeloma. Among the FDA approved and investigational CAR-T, primary refractory disease post CAR-T infusion is uncommon, however most patients eventually relapse. To gain insight to the myeloma cell and immune cell characteristics associated with early relapse (PD) versus durable response (DR), we examined myeloma cells and immune cells in both the bone marrow and blood by single cell RNA-seq. Method: Bone marrow aspirate and blood samples were collected prior to BCMA targeted CAR-T therapy. CD138 + and CD138 neg cells from bone marrow (BM) and peripheral blood mononuclear cells (PBMNC) were collected for 5' based scRNA-seq (10X Genomics). CD138 + cells were clustered by Harmony integration. Top expressing genes in unique clusters for PD and DR were analyzed by Ingenuity Pathway Analyses. To profile CD138 neg BM cells and PBMNC from myeloma patients, the scRNA-seq dataset was mapped to the human PBMC reference using the Seurat v4 reference-guiding approach. Differential gene expression between responders and non-responders was performed using the FindMarkers function for each of the immune cell clusters in the BM and PB. Results: BM and PB samples from 15 patients were analyzed. Among these, 5 patients had relapsed disease more than 1-year post CAR-T infusion (Durable Response, DR), and 10 patients had relapse within 1 year (early relapse, PD). Among these, CD138+ cells were available from 3 DR and 4 PD for analysis. Multimodal expression of BCMA were seen among the CD138+ cells across all samples. Using the mode of the highest expression level as a cut-off, patients with DR had a trend toward having higher fraction of BCMA high CD138+ cells compared to pts with PD. Clustering of all CD138+ cells by Harmony integration identified 12 distinct clusters, among which two clusters were unique to pts in DR, and one was unique to patients in PD. Ingenuity pathway analysis identified the top marker genes in these 3 clusters to be enriched in pathways for IL-15 signaling, BCR signaling, and primary immunodeficiency signaling. (Figure 1A-E.) Differential gene expression for immune cell clusters from CD138 neg BM cells and PBMNC were compared between pts in PD and DR. Interestingly, different patterns were seen in immune cells in PB and BM (Figure 1G). For example, compared to pts in PD, those with DR had decreased signaling in CD16 PB monocytes for interferon signaling and differentiation to macrophages, whereas the CD16 monocytes in the BM in pts in DR had increased expression of mitochondrial genes for anti-oxidant stress, polarization to M1 macrophage and anti-apoptosis. Similarly, pts in DR had CD8 Tcm in the BM with increased expression of genes involved in cell adhesion and anti-apoptosis, and CD4 Tcm in the BM with increased expression of genes in protein metabolism. Conclusion: scRNAseq analysis of myeloma cells suggest different tumor transcriptome profile may be identified in myeloma cells that could relapse early after CAR-T therapy. In addition, host immune profile both in the BM microenvironment and in systemic PB circulation could be associated with durable clinical response. Figure 1 Figure 1. Disclosures Kapoor: Pharmacyclics: Consultancy; Glaxo SmithKline: Research Funding; Amgen: Research Funding; Sanofi: Research Funding; AbbVie: Research Funding; Ichnos Sciences: Research Funding; Sanofi: Consultancy; Regeneron Pharmaceuticals: Research Funding; Takeda: Research Funding; Karyopharm: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy. Dingli: GSK: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Novartis: Research Funding; Apellis: Consultancy; Alexion: Consultancy. Kumar: Bluebird Bio: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Novartis: Research Funding; BMS: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Oncopeptides: Consultancy; Beigene: Consultancy; Amgen: Consultancy, Research Funding; Merck: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Lin: Merck: Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Takeda: Research Funding; Bluebird Bio: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sorrento: Consultancy; Juno: Consultancy; Legend: Consultancy; Gamida Cell: Consultancy; Vineti: Consultancy; Novartis: Consultancy; Janssen: Consultancy, Research Funding.

Respiratory tissue resident–like CD8 + T cells are associated with chronic lung sequelae post-acute COVID-19.

Abstract Purpose: Outcomes for resistant metastatic castration-resistant prostate cancer (CRPC) are poor. Stereotactic ablative radiotherapy (SABR) induces antitumor immunity in clinical and preclinical studies, but immunologic biomarkers are lacking. Patients and Methods: Eighty-nine patients with oligometastatic CRPC were identified by 11C-Choline-PET (Choline-PET) from August 2016 to December 2019 and treated with SABR. Prespecified coprimary endpoints were 2-year overall survival (OS) and PSA progression. Secondary endpoints included 2-year SABR-treated local failure and 6-month adverse events. Correlative studies included peripheral blood T-cell subpopulations before and after SABR. Results: 128 lesions in 89 patients were included in this analysis. Median OS was 29.3 months, and 1- and 2-year OS were 96% and 80%, respectively. PSA PFS was 40% at 1 year and 21% at 2 years. Local PFS was 84.4% and 75.3% at 1 and 2 years, respectively, and no grade ≥3 AEs were observed. Baseline high levels of tumor-reactive T cells (TTR; CD8+CD11ahigh) predicted superior local, PSA, and distant PFS. Baseline high levels of effector memory T cells (TEM; CCR7−CD45RA−) were associated with improved PSA PFS. An increase in TTR at day 14 from baseline was associated with superior OS. Conclusions: This is the first comprehensive effector T-cell immunophenotype analysis in a phase II trial before and after SABR in CRPC. Results are favorable and support the incorporation of immune-based markers in the design of future randomized trials in patients with oligometastatic CRPC treated with SABR.

Novel therapeutic strategies are needed for the treatment of rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. By using a combination of cell surface proteomics and transcriptomic profiling of RMS and normal muscle, we generated a catalog of targetable cell surface proteins enriched in RMS tumors. Among the top candidates, we identified B7-H3 as the major immunoregulatory molecule expressed by RMS tumors. By using a large cohort of tissue specimens, we demonstrated that B7-H3 is expressed in a majority of RMS tumors while not detected in normal human tissues. Through a deconvolution analysis of the RMS tumor RNA-seq data, we showed that B7-H3-rich tumors are enriched in macrophages M1, NK cells, and depleted in CD8+-T cells. Furthermore, in vitro functional assays showed that B7-H3 knockout in RMS tumor cells increases T-cell mediated cytotoxicity. Altogether, our study uncovers new potential targets for the treatment of RMS and provides the first biological insights into the role of B7-H3 in RMS biology, paving the way for the development of next-generation immunotherapies.