PD-L1 Positive Tumor Cells Research Articles

A preliminary study on the diagnostic performance of the uPath PD-L1 (SP263) artificial intelligence (AI) algorithm in patients with NSCLC treated with PD-1/PD-L1 checkpoint blockade.

The uPath PD-L1 (SP263) is an AI-based platform designed to aid pathologists in identifying and quantifying PD-L1 positive tumor cells in non-small cell lung cancer (NSCLC) samples stained with the SP263 assay. In this preliminary study, we explored the diagnostic performance of the uPath PD-L1 algorithm in defining PD-L1 tumor proportion score (TPS) and predict clinical outcomes in a series of patients with advanced stage NSCLC treated with single agent PD-1/PD-L1 checkpoint blockade previously assessed with the SP263 assay in clinical practice. 44 patients treated from August 2015 to January 2019 were included, with baseline PD-L1 TPS of ≥ 50%, 1-49% and < 1% in 38.6%, 25.0% and 36.4%, respectively. The median uPath PD-L1 score was 6 with a significant correlation with the baseline PD-L1 TPS (r: 0.83, p < 0.01). However, only 27 cases (61.4%) were scored within the same clinically relevant range of expression (≥ vs < 50%). In the study population the baseline PD-L1 TPS was not significantly associated with clinical outcomes, while the uPath PD-L1 score showed a good diagnostic ability for the risk of death at the ROC curve analysis [AUC: 0.81 (95%CI: 0.66-0.91), optimal cut-off of ≥ 3.2], resulting in 19 patients (43.2%) being u-Path low and 25 patients (56.8%) being uPath high. The objective response rate in uPath high and low was 51.6% and 25.0% (p = 0.1), respectively, although the uPath was significantly associated with overall survival (OS, HR 2.45, 95%CI: 1.19-5.05) and progression free survival (PFS, HR 3.04, 95%CI: 1.51-6.14). At the inverse probability of treatment weighting analysis used to balance baseline covariates, the uPath categories confirmed to be independently associated with OS and PFS. This preliminary analysis suggests that AI-based, digital pathology tools such as uPath PD-L1 (SP263) can be used to optimize already available biomarkers for immune-oncology treatment in patients with NSCLC.

Abstract 1048: Loss of MTAP expression is strongly linked to homozygous 9p21 deletion, unfavorable tumor phenotype and non-inflamed microenvironment in urothelial bladder cancer

Abstract 9p21 deletions are common in urothelial carcinoma and mostly include S-methyl-5′-thioadenosine phosphorylase (MTAP) a critical enzyme for DNA and RNA synthesis. Homozygous MTAP deletions result in a complete loss of expression which can be visualized by immunohistochemistry (IHC). Evidence is emerging that MTAP deficiency results in a critical vulnerability of cancer cells towards drugs targeting multiple different pathways that depend on a functional MTAP gene and causes resistance to immune checkpoint inhibitors. To determine the prevalence and clinical significance of MTAP deficiency and its relationship with the tumor microenvironment in urothelial bladder carcinomas, more than 2,500 tumors were analyzed by fluorescence in-situ hybridization (FISH) and IHC in a tissue microarray format, and the data were compared with existing data on intratumoral lymphocyte subsets and PD-L1 expression. The cohort included 1,826 patients that underwent radical cystectomy for muscle-invasive disease (pT2-4). 9p21 deletion was homozygous in 364 (21.3%) and heterozygous in 334 (19.5%) of 1,711 analyzable tumors. The rate of 9p21 deleted tumors increased from pTa G2 low (9.2% homozygous, 25.8% heterozygous) to pTa G2 high (32.6%, 20.9%; p&amp;lt;0.0001) but was slightly lower in pTa G3 (16.7% each). In pT2-pT4 carcinomas, 9p21 deletions were homozygous in 23.3% and heterozygous in 17.9%. Within muscle-invasive cancers, 9p21 deletions were tied to high pT (p=0.0014) and poor overall survival (p=0.0461). MTAP expression loss was strongly linked to homozygous 9p21 deletions. Complete absence of MTAP staining was seen in 96.1% of homozygous deleted cancers while only 4 of 249 MTAP negative cancers had retained at least one 9p21 copy (p&amp;lt;0.0001). Accordingly, absence of MTAP staining was also linked to advanced pT status and poor overall survival (p&amp;lt;0.05 each). Tumors with heterozygous 9p21 deletion did often show preserved but low-level expression of MTAP. 9p21 deletions were associated with low numbers of PD-L1 positive tumor cells (p&amp;lt;0.0001), CD8 (p=0.01) and CD4 positive lymphocytes (p=0.009), M2-macrophages (p&amp;lt;0.0001), and dendritic cells (p=0.0007). A similar tendency was observed for MTAP expression loss, but the level of significance was only reached for PD-L1 positive tumor cells (p&amp;lt;0.0001), M2 macrophages (p=0.001) and dendritic cells (p=0.012). Complete MTAP expression loss is common in urinary bladder cancer and strongly tied to homozygous 9p21 loss, aggressive disease, and non-inflamed microenvironment. Drugs targeting MTAP-deficiency may be highly useful in bladder cancer. MTAP IHC is an excellent surrogate for the detection of MTAP deficiency in this tumor entity. Citation Format: Natalia Gorbokon, Niklas Wößner, Viktoria Ahlburg, Simon Schallenberg, Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberger, Paul G. Bruch, Florian Roßner, Sefer Elezkurtaj, Maximilian Lennartz, Niclas C. Blessin, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Steffen Hallmann, Stefan Koch, Nico Adamini, Sarah Minner, Ronald Simon, Guido Sauter, Henrik Zecha, David Horst, Tobias Klatte, Thorsten Schlomm, Martina Kluth. Loss of MTAP expression is strongly linked to homozygous 9p21 deletion, unfavorable tumor phenotype and non-inflamed microenvironment in urothelial bladder cancer [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 1048.

Abstract 6189: Deep-learning model trained on multiplex immunofluorescence-stained tissue samples predicts the survival of patients with non-small cell lung cancer better than PD-L1 TPS alone

Abstract In non-small cell lung cancer (NSCLC), the composition and structure of the tumor immune microenvironment (TME) critically impact patient outcomes, necessitating improved computational approaches to characterize the TME beyond traditional metrics like the PD-L1 tumor proportion score (TPS). Here, we developed an interpretable deep-learning model that robustly predicts patient overall survival (OS) and identifies unique PD-L1 enriched neighborhoods linked to patient prognosis. We utilized a dataset of 507 NSCLC patient biopsy samples prospectively acquired as part of the DFCI ImmunoPROFILE project. Each sample was stained for Cytokeratin as tumor marker, PD-L1, PD-1, CD8, and FOXP3 using a targeted multiplex immunofluorescence assay. Inner tumor regions of interest were automatically processed for spatially resolved identification and quantification of marker expression at the single-cell level. The dataset was divided into 380 patients for model development and 127 for testing. The deep-learning model, a graph neural network (GNN), was trained to predict OS based on local cell neighborhoods using a weakly supervised training paradigm. The GNN models neighborhoods as a graph, with cells and their corresponding marker expression representing nodes and edges connecting adjacent cells, for an average of 42 cells per neighborhood. Survival predictions were obtained by averaging predictions over all neighborhoods from one sample. The GNN model demonstrated robust performance with a concordance index (c-index) of 0.79 on the test data, surpassing traditional metrics such as TPS and immune marker density-based models (c-index: 0.50 and 0.74, respectively). Beyond assessing performance, we investigated the features learned by the GNN using k-means clustering in the model’s feature space. We identified three clusters that were notably enriched in PD-L1 positive tumor cells. The first cluster shows an especially “high TPS” phenotype, where the GNN predicted such neighborhoods to be generally poor for survival. The other two clusters were additionally enriched with PD-L1 positive immune cells (ICs) and associated with favorable survival predictions. Notably, one cluster was also enriched with other types of ICs (”PD-L1 IC mixed”), which the GNN predicted as especially favorable for survival compared to exclusive enrichment of PD-L1 ICs (”PD-L1 IC dominant”). A score summarizing these three PD-L1 expression patterns was significantly associated with OS (p-value: 0.01; HR: 0.72, 95% CI: 0.16-1.27), validating the features learned by the GNN. In sum, we developed a highly interpretable deep-learning model leveraging cellular neighborhood graphs, which effectively identifies expression patterns correlating with NSCLC prognosis, offering promise for developing personalized treatment strategies. Citation Format: Katharina Viktoria Hoebel, James R. Lindsay, Joao V. Alessi, Jason L. Weirather, Ian D. Dryg, Jennifer Altreuter, Mark M. Awad, Scott J. Rodig, William E. Lotter. Deep-learning model trained on multiplex immunofluorescence-stained tissue samples predicts the survival of patients with non-small cell lung cancer better than PD-L1 TPS alone [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 6189.

Abstract 1807: CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ tumors

Abstract Introduction: CD137 (HGNC:TNFRSF9, 4-1BB) is a TNF receptor superfamily costimulatory receptor expressed by T cells and NK cells. Upon engagement, CD137 enhances immune cell survival, proliferation, cytokine production and memory formation. CD137 agonists in the clinic have shown tantalizing anti-tumor effects, however, hepatic toxicity has hampered clinical utility. To circumvent this limitation, we developed CB307, a half-life extended heavy-chain only Humabody® that selectively agonizes CD137 only in the presence of prostate specific membrane antigen (PSMA, HGNC:FOLH1). PSMA is highly upregulated on tumor cells in prostate cancer and tumor neovasculature in other solid tumors including lung (NSCLC), kidney (clear cell RCC), bladder and colorectal cancers. CB307 enhances immune cell activation in a PSMA-dependent manner. We present here preclinical mechanistic differentiators of CB307 with respect to non-targeted CD137 agonists. Methods: CD137 agonism, immune cell activation and cytokine secretion were evaluated in vitro using coculture assays in the presence/absence of PSMA-expressing tumor cells and in vivo with a transgenic mouse syngeneic tumor model. The ability of CB307 or non-targeted CD137 agonists to enhance tumor cell killing was additionally characterized in 3D spheroid cocultures. In vitro cocultures were used to determine the impact of PSMA expression level on CB307 activity. CB307 was evaluated as a single agent and in combination with PD-1 or PD-L1 inhibitors; tumoral checkpoint contribution was investigated using isogenic PD-L1 positive vs. PD-L1 CRISPR-deleted tumor cells. Results: CB307 or PD-1/PD-L1 inhibition alone induced IL-2 secretion in PBMC/PSMA-expressing tumor cell cocultures; this effect was synergistically enhanced when CB307 was combined with either PD-1 or PD-L1 inhibition. CB307 augmented tumor cell killing in 3D spheroids containing PSMA-expressing cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB307 in combination with PD-1/PD-L1 inhibition. In in vivo pharmacology studies in a syngeneic mouse model, CB307 demonstrated statistically significant inhibition of tumor growth versus control without peripheral T cell activation - consistent with pharmacology restricted to the PSMA+ tumor microenvironment, whereas an anti-CD137 monoclonal antibody induced tumor growth inhibition accompanied by peripheral immune cell proliferation and cytokine production. Summary: CB307 is a novel CD137 agonist that selectively enhances immune cell activity only in the presence of PSMA-positive cells. Our data demonstrate PSMA-targeted CB307 immunological pharmacology both in vitro and in vivo which has supported the ongoing Phase 1 ‘POTENTIA’ clinical trial (NCT04839991). Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Clare Song, Anca-Giuilia Cionca, Joanne Fernando, Phillip D. Bartlett, Philip Bland-Ward. CB307: a novel targeted CD137 agonist for enhancement of immune cell responses to PSMA+ 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 1807.

MP14-15 MACROPHAGES AND REGULATORY T-CELLS AS PROGNOSTIC FACTORS IN PATIENTS WITH UROTHELIAL MUSCLE-INVASIVE BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP14-15 MACROPHAGES AND REGULATORY T-CELLS AS PROGNOSTIC FACTORS IN PATIENTS WITH UROTHELIAL MUSCLE-INVASIVE BLADDER CANCER Florestan Koll, Severine Banek, Luis Kluth, Jens Köllermann, Andreas Weigert, Felix Chun, Peter Wild, and Henning Reis Florestan KollFlorestan Koll More articles by this author , Severine BanekSeverine Banek More articles by this author , Luis KluthLuis Kluth More articles by this author , Jens KöllermannJens Köllermann More articles by this author , Andreas WeigertAndreas Weigert More articles by this author , Felix ChunFelix Chun More articles by this author , Peter WildPeter Wild More articles by this author , and Henning ReisHenning Reis More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The prognosis of muscle-invasive urothelial bladder cancer (MIBC) is poor and response rates to systemic therapy are insufficient. Infiltration of tumors by immune cells has been described to be associated with response rates to immune- and chemotherapy and patient’s outcome. However, tumor-associated macrophages (TAMs) are abundant in many tumors and can promote cancer progression. In this study we evaluate different subsets of immune cells in the tumor microenvironment (TME) as predictors of prognosis in MIBC and survival rates with adjuvant chemotherapy. METHODS: We used multiplex immunohistochemistry (IHC) to quantify and characterize immune, tumor and stroma cells: double negative T-cells (CD3+CD4-CD8-); T-helper cells (CD4+); cytotoxic T-cells (CD8+); PD-1 positive T-cells; macrophages (CD163+); regulatory T-cells (Tregs; FoxP3+)); fibroblasts (Vimentin+); immune cells (CD45+); tumor cells (panCK+); PD-L1 positive tumor cells; proliferating immune cells (CD45+Ki67+); proliferating tumor cells (panCK+Ki67+). Analysis was performed in 101 MIBC patients receiving radical cystectomy. Uni- and multivariate Cox regression were used to identify cell types that predict prognosis. Patients were divided into three clusters for their macrophage and Treg infiltration. RESULTS: High Treg infiltration was associated with better overall survival (OS) (HR 0.1, 95% CI 0.01-0.7; p=0.03) and high macrophage infiltration was associated with decreased OS (HR 10.9, 95% CI 2.8-40.5; p=0.0004) in the multivariate Cox-regression model adjusting for tumor stage, lymph node status and application of adjuvant chemotherapy. The cluster with high Treg concentration was also enriched for other immune cells and cells showed high PD1 and PD-L1 expression. Patients in the cluster with high macrophage concentration had the worst OS also when adjuvant chemotherapy was given. We validated the findings using standardized IHC for CD163 as a macrophage marker using a digital analysis software in 141 patients. CONCLUSIONS: In MIBC high macrophage concentration is an independent predictor of poor prognosis. High levels of Tregs are associated with other immune cells in the TME which might lead to better survival rates. Using routine IHC for TAMs (CD163) should be prospectively validated to confirm findings and to evaluate the predictive value for response to systemic therapies. Source of Funding: Deutsche Krebshilfe (German Cancer Aid). Wilhelm-Sander Foundation. Hessen State Ministry for Higher Education, Research and the Arts © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e188 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Florestan Koll More articles by this author Severine Banek More articles by this author Luis Kluth More articles by this author Jens Köllermann More articles by this author Andreas Weigert More articles by this author Felix Chun More articles by this author Peter Wild More articles by this author Henning Reis More articles by this author Expand All Advertisement PDF downloadLoading ...

Tumor cell HLA class I expression and pathologic response following neoadjuvant immunotherapy for newly diagnosed head and neck cancer

ObjectivesBiomarkers are needed to identify patients likely to respond to neoadjuvant immunotherapy (NIT) prior to receiving definitive treatment. Materials and MethodsWe hypothesized that expression of tumor cell HLA class I would correlate with pathologic response (PR) following NIT for primary untreated head and neck cancer. Multispectral immunofluorescence of pre- and post-treatment biopsy specimens from a neoadjuvant study of bintrafusp alfa, a dual TGF-β and PD-L1 inhibitor, was performed. ResultsDiscordant expression of tumor cell HLA class I and PD-L1 measured by multispectral immunofluorescence was observed with most positive tumor cells expressing HLA class I or PD-L1 but not both. Spatial analysis revealed colocalization between tumor parenchyma T cells and HLA class I positive tumors cells, but no clear colocalization between T cells and PD-L1 positive tumor cells. Greater pre-treatment tumor cell HLA class I expression, but not PD-L1 expression or tumor T cell infiltration, correlated with the development of a PR. Additionally, increased tumor cell HLA class I expression after NIT compared to before NIT correlated with development of a PR, whereas inconsistent changes in PD-L1 and T cell infiltration were observed after treatment in all patients. ConclusionsThese data provide the rationale for the study of tumor cell HLA class I expression in larger prospective studies powered to determine the performance of biomarkers of PR in newly diagnosed HNSCC patients receiving NIT.

The redirected killing of PD-L1 positive tumor cells by the expanded mucosa-associated invariant T (MAIT) cells is mediated with a bispecific antibody targeting TCR Vα7.2 and PD-L1

Pan-T cell targeting by CD3-based T cell engagers has brought program-shift treatment and management of blood tumors. However, these modalities have been shown to provoke all types of T cells leading to cytokine storm syndrome, and activate Treg cells. Thus, modulating and potentiating the antitumor responses of a specific T cell subset was encouraged. We initially found that high purity of mucosa-associated invariant T (MAIT) cells could be expanded by the combination of plate-immobilized Vα7.2 mAb (Clone 3C10) and IL2 plus IL15. Then, we generated a novel anti-Vα7.2 TCR bsAb, Vα7.2 x PD-L1, to invoke the anti-tumor potency of these expanded MAIT cells. Furthermore, our data have demonstrated that Vα7.2 x PD-L1 could mediate the cell-to-cell conjunction between MAIT cell and tumor cell line, selectively elicit the activation, cytokine production, degranulation, and cytotoxicity of the expanded MAIT cells in the presence of target cell only. Collectively, this proof-of-concept study provides a new tool to explore the clinical potential of MAIT cells in fighting against PD-L1 positive solid tumors and suggests additional encouragement in designing novel T cell engagers targeting TCR alpha chain specific innate-like T cells subsets, other than pan CD3+ T cells.

ANK Therapeutic Prospects and Usefulness of PD-L1 and NK Activity as Biomarkers for Predicting Treatment Efficacy Revealed from the Treatment Course of Patients with HTLV-1-Associated Bronchioloalveolar Disease

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm with poor prognosis that can present as HTLV-1associated bronchioloalveolar disease (HABA). Chemotherapy is recommended for ATL; however, it is not very effective against all types of ATL. Furthermore, there are no effective treatments for smoldering HABA-associated ATL. We present a case in which amplified natural killer cell (ANK) therapy was effective in a woman in her early 80s who was previously diagnosed with ATL-related smoldering HABA and presented with dyspnea and productive cough on exertion. The symptoms were suppressed for approximately 10 months after the first treatment, but then gradually worsened. About a year later, a second treatment was followed by mild side effects. Suppression of ATL cell proliferation by repeated doses of ANK therapy appears to be effective in this patient. The therapeutic effect was high even with long treatment intervals, and the efficacy and safety of repeated treatments have been demonstrated. ANK therapy is expected to be the mainstay of treatment ATL and HABA. ANK therapy has been reported to kill PD-L1 positive tumor cells and some solid tumors with excellent responses have many PDL1-positive tumor cells. ANK therapy is thought to be effective for ATL because there are many PD-L1-positive tumor cells. Furthermore, administration of activated NK cells may increase tumor-killing activity in those patients with reduced NK activity. While future studies are needed, PD-L1 positive rate and NK activity may be biomarkers for the effectiveness of ANK therapy.

ANK Therapeutic Prospects and Usefulness of PD-L1 and NK Activity as Biomarkers for Predicting Treatment Efficacy Revealed from the Treatment Course of Patients with HTLV-1-Associated Bronchioloalveolar Disease

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm with poor prognosis that can present as HTLV-1associated bronchioloalveolar disease (HABA). Chemotherapy is recommended for ATL; however, it is not very effective against all types of ATL. Furthermore, there are no effective treatments for smoldering HABA-associated ATL. We present a case in which amplified natural killer cell (ANK) therapy was effective in a woman in her early 80s who was previously diagnosed with ATL-related smoldering HABA and presented with dyspnea and productive cough on exertion. The symptoms were suppressed for approximately 10 months after the first treatment, but then gradually worsened. About a year later, a second treatment was followed by mild side effects. Suppression of ATL cell proliferation by repeated doses of ANK therapy appears to be effective in this patient. The therapeutic effect was high even with long treatment intervals, and the efficacy and safety of repeated treatments have been demonstrated. ANK therapy is expected to be the mainstay of treatment ATL and HABA. ANK therapy has been reported to kill PD-L1 positive tumor cells and some solid tumors with excellent responses have many PDL1-positive tumor cells. ANK therapy is thought to be effective for ATL because there are many PD-L1-positive tumor cells. Furthermore, administration of activated NK cells may increase tumor-killing activity in those patients with reduced NK activity. While future studies are needed, PD-L1 positive rate and NK activity may be biomarkers for the effectiveness of ANK therapy.

Vγ2 x PD-L1, a Bispecific Antibody Targeting Both the Vγ2 TCR and PD-L1, Improves the Anti-Tumor Response of Vγ2Vδ2 T Cell.

The potent cytotoxic property of Vγ2Vδ2 T cells makes them attractive for adoptive T cell transfer therapy. The transfusing of the expanded Vγ2Vδ2 T cells into cancer patients shows well-tolerated, but the clinical response rates are required to be improved, implying that there is still an unmet efficacy with low toxicity for this novel anti-tumor therapy. In this study, we test the anti-tumor efficacy of a Y-body-based bispecific antibody (bsAb) Vγ2 x PD-L1 that preferentially redirects Vγ2Vδ2 T cells to combat PD-L1 positive tumor cells. With nanomolar affinity levels to Vγ2Vδ2 T cells and PD-L1+ tumor cells, Vγ2 x PD-L1 bridges a Vγ2Vδ2 T cell with a SKOV3 tumor cell to form a cell-to-cell conjugation. In a PD-L1-dependent manner, the bsAb elicits effective activation (CD25+CD69+), IFNγ releasing, degranulation (CD107a+), and cytokine production (IFNγ+ and TNFα+) of expanded Vγ2Vδ2 T cells. The activations of the Vγ2Vδ2 T cells eliminate PD-L1-expressing human cancer cell lines, including H1975, SKOV3, A375, H1299, and H2228 cells, but not PD-L1 negative cells including HEK-293 (293) cells and healthy PBMCs. Finally, we show that combining Vγ2 x PD-L1 with adoptively transferring Vγ2Vδ2 T cells inhibits the growth of existing tumor xenografts and increases the number of Vγ2Vδ2 T cells into the tumor bed. Vγ2 x PD-L1 represents a promising reagent for increasing the efficacy of adoptively transferred Vγ2Vδ2 T cells in the treatment of PD-L1 positive malignant tumors.

Abstract 3543: Altering tumor immunophenotypes with PD-L1 engineered toxin bodies

Abstract Targeting of PD-L1 checkpoint has shown clinical efficacy in multiple solid tumor indications. Currently approved PD-L1 targeted approaches rely on the blocking activity of monoclonal antibodies (mAbs) which sterically inhibit PD-L1 thus preventing PD-1 mediated immune checkpoint activity. Although these mAbs have shown activity in the clinic, the need for pre-existing tumor specific immunity and tumor immune infiltration precludes responses in some patients and leads to resistance in others. Therefore, there remains a need for new modalities and treatment paradigms. Molecular Templates has developed MT-6402, an engineered toxin body (ETB) targeting PD-L1 designed to overcome the challenges of current PD-L1 targeting approaches by 1) directly depleting PD-L1 positive tumor or immunosuppressive immune cells displaying PD-L1 and 2) alteration of the tumor immunophenotype through the cytoplasmic delivery of an HLA:A*02 restricted cytomegalovirus (CMV) antigen. Delivery of this antigen is meant to leverage recruitment of existing CMV-restricted cytotoxic T lymphocytes (CTLs) for cell-mediated cytotoxic depletion (antigen seeding technology, AST) of CMV antigen presenting tumor cells. MT-6402 is currently in a phase I open-label, dose escalation and expansion study in subjects with advanced solid cancers that express PD-L1 (NCT04795713). Initial assessment of pharmacodynamic markers for PD-L1 and CMV-mediated ETB activity in a subject with confirmed PD-L1+ tumor cells, CMV positivity, and HLA:A*02 restriction has shown serum phenotypes associated with robust checkpoint inhibitor activity and full extravasation of circulating CMV-specific T cells. In addition to assessing early clinical data from the HLA: A*02 restricted MT-6402 trials, expansion of ETB AST to a broader patient population requires testing of ETBs engineered with the ability to deliver CMV antigens across a broad range of HLA restriction, including HLA:A*01, HLA:A*03, and HLA:A*24. To this end, ETBs were benchmarked against MT-6402 to identify candidates that retain comparable specificity, selectivity, and activity. Substitution of peptide antigens did not alter specificity or selectivity of ETBs compared to MT-6402. Candidate ETB binding profiles and potency were comparable to MT-6402. ETBs delivered an antigen seeding response in a PD-L1 dependent and HLA specific manner. In vivo efficacy of candidate ETBs was comparable in a murine efficacy xenograft model with MT-6402. An ex vivo cytokine release assay in a co-culture setting using HLA matched CTLs and PD-L1+ target cells, treatment with AST capable ETBs resulted in secretion of antigen specific T cell mediated immune cytokines compared to AST null controls. These cytokines overlap with cytokine signatures observed after dosing MT-6402 in HLA:A*02 patients. Preclinical assessment of the safety profile of candidates is ongoing and further development is slated for 2021. Citation Format: Swati Khanna, Elizabeth M. Kapeel, Lauren R. Byrne, Elizabeth Saputra, Steven Rivera, Lindsey Aschenbach, Lilia A. Rabia, Garrett L. Cornelison, Rachael M. Orlandella, Brigitte Brieschke, Michaela Sousares, Jay Zhao, Garrett L. Robinson, Chris Moore, Joseph D. Dekker. Altering tumor immunophenotypes with PD-L1 engineered toxin bodies [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 3543.

Cellular engagement and interaction in the tumor microenvironment predict non-response to PD-1/PD-L1 inhibitors in metastatic non-small cell lung cancer

Immune checkpoint inhibitors (ICI) with anti-PD-1/PD-L1 agents have improved the survival of patients with metastatic non-small cell lung cancer (mNSCLC). Tumor PD-L1 expression is an imperfect biomarker as it does not capture the complex interactions between constituents of the tumor microenvironment (TME). Using multiplex fluorescent immunohistochemistry (mfIHC), we modeled the TME to study the influence of cellular distribution and engagement on response to ICI in mNSCLC. We performed mfIHC on pretreatment tissue from patients with mNSCLC who received ICI. We used primary antibodies against CD3, CD8, CD163, PD-L1, pancytokeratin, and FOXP3; simple and complex phenotyping as well as spatial analyses was performed. We analyzed 68 distinct samples from 52 patients with mNSCLC. Patients were 39–79 years old (median 67); 44% were male and 75% had adenocarcinoma histology. The most used ICI was atezolizumab (48%). The percentage of PD-L1 positive epithelial tumor cells (EC), degree of cytotoxic T lymphocyte (CTL) engagement with EC, and degree of CTL engagement with helper T lymphocytes (HTL) were significantly lower in non-responders versus responders (p = 0.0163, p = 0.0026 and p = 0.0006, respectively). The combination of these 3 characteristics generated the best sensitivity and specificity to predict non-response to ICI and was also associated with shortened overall survival (p = 0.0271). The combination of low CTL engagement with EC and HTL along with low expression of EC PD-L1 represents a state of impaired endogenous immune reactivity. Together, they more precisely identified non-responders to ICI compared to PD-L1 alone and illustrate the importance of cellular interactions in the TME.

Immunohistochemical assessment of PD-L1 expression using three different monoclonal antibodies in triple negative breast cancer patients

BackgroundPD-L1 receptor expression in breast cancer tissue can be assessed with different anti-human PD-L1 monoclonal antibodies. The performance of three specific monoclonal antibodies in a head-to-head comparison is unknown. In addition, a potential correlation of PD-L1 expression and clinico-pathological parameters has not been investigated.MethodsThis was a retrospective study on tissue samples of patients with histologically confirmed triple negative breast cancer (TNBC). PD-L1 receptors were immune histochemically stained with three anti-human PD-L1 monoclonal antibodies: 22C3 and 28-8 for staining of tumor cell membranes (TC) and cytoplasm (Cyt), SP142 for immune cell staining (IC). Three different tissue samples of each patient were evaluated separately by two observers in a blinded fashion. The percentage of PD-L1 positive tumor cells in relation to the total number of tumor cells was determined. For antibodies 22C3 and 28-8 PD-L1 staining of 0 to < 1% of tumor cells was rated "negative", 1–50% was rated "positive" and > 50% was rated "strong positive". Cyt staining was defined as “negative” when no signal was observed and as “positive”, when any positive signal was observed. For IC staining with SP142 all samples with PD-L1 expression ≥ 1% were rated as “positive”. Finally, the relationship between PD-L1 expression and clinico-pathological parameters was analyzed.ResultsTissue samples from 59 of 60 enrolled patients could be analyzed. Mean age was 55 years. Both the monoclonal antibodies 22C3 and 28-8 had similar properties, and were positive for both TC in 13 patients (22%) and for Cyt staining in 24 patients (40.7%). IC staining with antibody SP142 was positive in 24 patients (40.7%), who were also positive for Cyt staining. The differences between TC and Cyt staining and TC and IC staining were significant (p = 0.001). Cases with positive TC staining showed higher Ki67 expression compared to those with negative staining, 40 vs 30%, respectively (p = 0.05). None of the other clinico-pathological parameters showed any correlation with PDL1 expression.ConclusionsAntibodies 22C3 and 28-8 can be used interchangeably for PD-L1 determination in tumor cells of TNBC patients. Results for Cyt staining with 22C3 or 28-8 and IC staining with SP142 were identical. In our study PD-L1 expression correlates with Ki67 expression but not with OS or DFS.

Clinicopathological correlation and prognostic value of PD-L1 expression in renal cell carcinoma

Introduction: The expression of programmed cell death-ligand 1 (PD-L1) and its correlation with the prognosis of renal cell carcinoma (RCC) remains controversial. Objectives: This study aimed to study PD-L1 expression in tumor cells and tumor-infiltrating lymphocytes (TILs) in patients with RCC and its association with clinicopathological factors and survival outcomes. Patients and Methods: PD-L1 expression in tumor cells and TILs was analyzed using immunohistochemistry (IHC) from patients with histologically proven RCC. Results: PD-L1 was positive in tumor cells for 55.8% of patients. PDL-1 expression in TIL was reported in 31.2 % of patients. Patients with PDL1 positive tumor cells had higher median tumor size (P=0.07), higher nuclear grade (P=0.56), and higher lymphovascular invasion (LVI) (P=0.23). Patients with PDL1 positive TILs were significantly associated with larger median pathological tumor size (P=0.02), higher probability of renal fat invasion (P=0.001), higher nuclear grade (P=0.05), higher probability of positive margin (P=0.02), positive LVI (P=0.03), higher pathological T stage (P=0.0004); whereas patients with PDL-1 negative TILs had earlier stage at presentation (stage I-II) (P=0.004). There was no statistically significant difference in disease-free survival (DFS), progression-free survival (PFS), or overall survival (OS) for PD-L1 expression of tumor cells and TILs. Conclusion: PDL1 positivity in TILs and not in tumor cells was significantly associated with more aggressive features, and higher stage. No association was found with DFS, PFS, or OS. These data suggest that PD-L1 expression of TILs in RCC tumors contributes to cancer aggressiveness.

L’évaluation des mutations de l’EGFR des carcinomes non à petites cellules de stade précoce

AbstractThe new adjuvant and/or neoadjuvant therapies for early stage and resectable non-small cell lung carcinoma (NSCLC), such as the targeted therapies on activating the EGFR mutations and the immunotherapies or the immuno-chemotherapies, lead mandatory the assessment of different predictive biomarkers of the therapeutic efficacy. The decision making for the treatment is directly associated with the percentage of PD-L1 positive tumor cells and with the status of EGFR and ALK. These new therapeutic opportunities require many challenges to the pathology laboratories, notably the establishment of new decisional algorithms that need to be integrated in daily practice.

Immune and Epigenetic Landscape of TP53-mutated Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes (HR-MDS)

Immune and Epigenetic Landscape of TP53-mutated Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes (HR-MDS)

Dinitrophenol-mediated modulation of an anti-PD-L1 VHH for Fc-dependent effector functions and prolonged serum half-life

Single-domain antibodies, VHHs or nanobodies, represent a promising set of alternatives to conventional therapeutic antibodies, gaining substantial attention in the field of cancer immunotherapy. However, inherent drawbacks of nanobodies such as fast clearance from blood circulation and lack of immune effector functions often led to unsatisfactory therapeutic efficacy. We previously reported that dinitrophenyl modification of an anti-EGFR VHH conferred Fc-dependent immune effector functions and elongated serum half-life on it through recruiting of hapten antibodies, resulting in improved immunotherapy efficacy in vivo. In the present work, we further tested the versatility of this approach in the case of an anti-PD-L1 blockade VHH (KN035). Site-specific dinitrophenyl conjugation did not impair the binding capacity of KN035 portion to PD-L1, but indirectly restored its immune effector functions, manifested by the observed antibody dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement-dependent cytotoxicity against PD-L1 positive tumor cells. Significant delay of blood clearance of dinitrophenylated KN035 was evidenced by the prolonged half-life of ca. 22 h. This approach, using small hapten molecule conjugation, loaded additional antibody-mediated tumor killing mechanisms to PD-L1 blockade VHH and therefore improved efficacy is anticipated in the future in vivo therapeutic studies. Thus, our results underscore the power of this versatile approach for achieving desirable properties of VHH-based or similar therapeutics.

Abstract 1628: Engineered toxin bodies targeting PD-L1 to alter tumor immunophenotypes and deliver broad antigenic diversity and patient coverage

Abstract Targeting PD-L1 has shown clinical efficacy in multiple solid tumor indications. Currently approved PD-L1 targeted approaches rely on monoclonal antibodies which sterically inhibit PD-L1 and prevent PD-1 mediated checkpoint activity. While these molecules have shown great activity in the clinic, the need for pre-existing tumor specific immunity and immune infiltration precludes responses in some patients and leads to resistance in others. Therefore, there remains a need for new modalities and treatment paradigms in these indications. Molecular Templates has developed MT-6402, an engineered toxin body (ETB) targeting PD-L1, as a single agent immunotoxin designed to overcome the challenges of current PD-L1 targeting approaches by 1) directly depleting PD-L1 positive tumor cells or immunosuppressive immune cells displaying PD-L1 in the tumor microenvironment and 2) delivery of an HLA: A*02 restricted viral peptide to alter the tumor immunophenotype for recruitment of CMV-restricted CTLs to target the tumor for depletion (antigen seeding). MT-6402 is slated for clinical development in 2021. Here we describe the preclinical characterization of several ETB candidates derived from MT-6402 delivering antigenic peptides restricted to the most prevalent MHC haplotypes in the U.S. population to broaden the patient population suitable for antigen seeding. ETBs were engineered with the ability to deliver viral peptides across a range of HLA restriction, including HLA: A*01, HLA: A*03, and HLA: A*24. ETBs were screened and benchmarked against MT-6402 and candidates were identified that retain comparable specificity, selectivity, and potency. Alteration of peptide antigen did not change the specificity or selectivity of ETBs which retained similar PD-L1 binding profiles to MT-6402. Binding profiles correlated to targeted potency and ETBs with varied HLA restricted peptides were found to target tumor and immune cells for depletion with similar potency to MT-6402. ETBs delivered an antigen seeding response in a PD-L1 dependent manner and only in conditions in which tumor cell and CTLs shared a matched HLA to the delivered antigenic peptide specificity. Preclinical assessment of the in vivo efficacy and safety profile of candidates is ongoing and further development is slated for 2021. Citation Format: Joseph D. Dekker, Swati Khanna, Elizabeth Saputra, Wenzhao Dong, Lindsey Aschenbach, Lilia A. Rabia, Garrett L. Cornelison, Michaela Sousares, Jay Zhao, Garrett L. Robinson, Betty Chang, Hilario J. Ramos. Engineered toxin bodies targeting PD-L1 to alter tumor immunophenotypes and deliver broad antigenic diversity and patient coverage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1628.

Large scale multiomic analysis suggests mechanisms of resistance to immunotherapy in leiomyosarcoma.

11512 Background: Leiomyosarcomas (LMS) have been reported to have immunohistochemical (IHC) and gene expression signatures suggestive of an immune-responsive tumor microenvironment. Despite this, immune checkpoint inhibitors have demonstrated minimal activity in LMS. We examined molecular profiles of LMS specimens from multiple institutions to explore mechanisms of immunotherapy (IO) resistance. Methods: LMS specimens (n = 1115), including 701 uterine (uLMS) and 414 soft tissue site (stLMS) samples, underwent next-generation sequencing (NGS) of DNA (592-gene panel or whole exome) and RNA (whole transcriptome, n = 537) at Caris Life Sciences (Phoenix, AZ). A threshold of 10 mut/Mb was used to identify high tumor mutational burden (TMB-H). IHC was performed for PD-L1 (SP142; 2+|5% positive). Deficient mismatch repair (dMMR)/high microsatellite instability (MSI-H) was tested by IHC and NGS, respectively. RNA expression was analyzed using Gene Set Enrichment Analysis and Microenvironment Cell Populations-counter, with results compared to melanoma (n = 1255) as a representative immunogenic tumor type. P-values were adjusted for multiple hypothesis testing. Results: TMB-H was observed in 3.8% (n = 41) of LMS specimens, with a median of 5 mut/Mb (IQR 3.3-6.7). dMMR/MSI-H was rarely detected (1.5%, n = 17), whereas 8.2% (n = 88) were positive for PD-L1 expression. uLMS and stLMS did not differ in TMB-H (3.4 vs 4.5%, p = 0.277), PD-L1 expression (8.6 vs 7.4%, p = 0.322), or dMMR/MSI-H (2.0 vs 0.7% p = 0.207). stLMS demonstrated upregulation of immune-related gene sets, including interferon γ (p = 0.035) and α (p = 0.033) response, inflammatory response (p = 0.038), interleukin-6/STAT3 signaling (p = 0.030), and TNFα signaling (p = 0.026) compared to uLMS. Immune cell infiltration was increased in stLMS over uLMS, most notably for CD8 T-cell and B-cell abundance ( &gt; 2-fold increase, p &lt; 0.0001). Compared to melanoma, all LMS had lower abundance of CD8 T cells, cytotoxic lymphocytes, and B-cells ( &gt; 2-fold decrease, p &lt; 0.0001). Fibroblasts were more prevalent in LMS relative to melanoma (3.2-fold increase, p &lt; 0.0001). Interestingly, while higher CD8 T-cell infiltration was positively associated with dMMR/MSI-H among LMS specimens (p = 0.032), TMB-H and PD-L1 expression were associated with lower CD8 T-cell infiltration (p &lt; 0.01). Conclusions: Only a small proportion of LMS are TMB-H or MSI-H, suggesting that the neoantigen burden in LMS may be insufficient to promote a robust anti-tumor response, even in the presence of PD-L1 positive tumor cells. Traditional predictive biomarkers of response to IO are unlikely to be useful in LMS. Furthermore, both uLMS and stLMS have an immune microenvironment characterized by a high fibroblast and low T cell abundance relative to melanoma. Future IO trials in LMS should focus on combination therapies that may reverse the observed T-cell exclusion/desmoplastic phenotype.

Bispecific Antibody PD-L1 x CD3 Boosts the Anti-Tumor Potency of the Expanded Vγ2Vδ2 T Cells.

Vγ2Vδ2 T cell-based immunotherapy has benefited some patients in clinical trials, but the overall efficacy is low for solid tumor patients. In this study, a bispecific antibody against both PD-L1 and CD3 (PD-L1 x CD3), Y111, could efficiently bridge T cells and PD-L1 expressing tumor cells. The Y111 prompted fresh CD8+ T cell-mediated lysis of H358 cells, but spared this effect on the fresh Vδ2+ T cells enriched from the same donors, which suggested that Y111 could bypass the anti-tumor capacity of the fresh Vγ2Vδ2 T cells. As the adoptive transfer of the expanded Vγ2Vδ2 T cells was approved to be safe and well-tolerated in clinical trials, we hypothesized that the combination of the expanded Vγ2Vδ2 T cells with the Y111 would provide an alternative approach of immunotherapy. Y111 induced the activation of the expanded Vγ2Vδ2 T cells in a dose-dependent fashion in the presence of PD-L1 positive tumor cells. Moreover, Y111 increased the cytotoxicity of the expanded Vγ2Vδ2 T cells against various NSCLC-derived tumor cell lines with the releases of granzyme B, IFNγ, and TNFα in vitro. Meanwhile, the adoptive transferred Vγ2Vδ2 T cells together with the Y111 inhibited the growth of the established xenografts in NPG mice. Taken together, our data suggested a clinical potential for the adoptive transferring the Vγ2Vδ2 T cells with the Y111 to treat PD-L1 positive solid tumors.