Supplementary Figure S8: Correlation of immune infiltration (IHC) and gene expression profiles

Response to TIL cell therapy is associated with presence of tumor-reactive clonotypes in the infusion product (Chiffelle Immunity 2024, Dean SITC 2024). Incorporating 4-1BB agonism in the pre-rapid expansion protocol (pre-REP) phase of non-engineered TIL manufacturing has been shown to increase TIL expansion and enrich for putative tumor-specific T cells (Frank SITC 2021); TIL engineering and modifications of the REP process may further drive such enrichment. OBX-115 TIL are engineered with regulatable, membrane-bound interleukin 15 (mbIL15), a cytokine that supports expansion of memory CD8+ T cells (Zhang Immunity 1998) in a manufacturing process that includes 4-1BB agonism in both pre-REP and REP (Frank SITC 2021). We sought to determine the phenotype and tumor reactivity of OBX-115 TIL compared with conventional non-engineered TIL in NSCLC. TIL were generated from paired NSCLC tumor samples using either the OBX-115 process or an IL2-based conventional process. Tumor digests were sequenced using combined single-cell RNA & TCR sequencing. Using a validated tumor-reactive TIL gene-expression profile (Meng Sci Trans Med 2023, Lowery Science 2022), we computationally predicted putative tumor-reactive clonotypes in OBX-115 vs conventional non-engineered TIL. Bulk TCR Vbeta sequencing was used to track the dynamics of these clonotypes throughout TIL expansion. TIL were phenotyped via flow cytometry and assessed for functional reactivity against autologous tumor digests or patient tumor-derived cell lines (PDc) in 3D co-cultures. The OBX-115 process generated a greater number of pre-REP TIL per tumor fragment vs conventional process (average 1.4 × 107 vs 2.3 × 105 cells, p=0.04, n=7), indicative of manufacturing success. Post-REP, OBX-115 TIL displayed higher proportion of CD39-CD69- “stem-like” progenitor cells (Krishna Science 2020) (57±9.6% vs 22±9.0%, p=0.010) and trends toward increased proportion of CD8+ T cells (94±2% vs 57±37%, p=0.133) and lower proportion of PD-1+ cells (3.8±1.6% vs 17±9.8%, p=0.054), relative to conventional TIL. OBX-115 was enriched for putative tumor-reactive TCR clonotypes compared to the conventional TIL, even when sorted for CD8+ cells (tumor-reactive signature score [Meng Sci Trans Med 2023], p<0.05). Moreover, OBX-115 demonstrated superior functional tumor reactivity relative to conventional TIL, marked by increased cytotoxicity against autologous tumor cell lines in 3D co-cultures (p<0.05) and similar or higher IFNγ secretion upon co-culture with autologous tumor digests. Here, we demonstrate that the OBX-115 manufacturing process from NSCLC tumor tissue, incorporating cell-armored-mbIL15-driven expansion and 4-1BB agonism, generates higher TIL yield and a more stem-like phenotype than conventional non-engineered TIL process. Computational analysis reveals the OBX-115 process enriches for putative tumor-reactive TCR clonotypes, which is further supported by in vitro co-culture assays. Together, these favorable attributes support further investigation of OBX-115 in patients with metastatic NSCLC (NCT06060613). Adam J. Schoenfeld, Alonso Villasmil Ocando, Zheng Ao, Bulent A. Aksoy, Ngoc Ly, Nishita Roy, Anand Veerapathran, Giridharan Ramsingh, Jan ter Meulen, Michelle Ols, Matthew Bott. OBX-115 TIL from non-small cell lung cancer (NSCLC) are enriched for putative tumor-reactive, stem-like T cells with enhanced tumor cytotoxicity: Results from multimodal phenotyping analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB359.

9519 Background: Non-engineered TIL cell therapy is approved for ICI-resistant advanced melanoma, but requires co-administration of toxic high-dose IL2. OBX-115 engineered TIL express mbIL15 under pharmacologic regulation using the FDA-approved small-molecule drug acetazolamide (ACZ), abrogating the need for IL2. We present data supporting OBX-115 mechanism of action. Methods: Trial design and clinical results were previously reported (Amaria ASCO 2024; NCT05470283); briefly, pts received lymphodepletion (Day [D] -7 to -1) followed by OBX-115 infusion (D0) and ≤7 days of orally administered ACZ (D2–9). Peripheral blood (PB) and tumor tissue samples were collected for longitudinal ddPCR analysis and immune profiling. Results: Eight pts received OBX-115 (fresh) and are included in this analysis. PB samples demonstrated ACZ-driven OBX-115 TIL expansion, reaching a median of 1697 cells/µL at D14 (approximate day of OBX-115 expansion peak); in 3 pts with ≥6 mo follow-up, OBX‐115 remained detectable through 6 mo and ongoing up to 15 mo. In the immediate post-infusion phase (up to D14), PB flow cytometry indicated expansion of product-derived CD3+CD8+ cells expressing Ki67 (during ACZ exposure) and endogenous NK cells (CD3-CD56+), while CD4+ cell levels decreased (Table). Post-infusion tumor tissue demonstrated presence of IL15-expressing T cells (of CD3+: D21, 68.6%; D42, 88.4%). Importantly, median post-infusion serum levels of IL15 and IL7 were not significantly elevated above Baseline through D42 (paired one-tailed t-test adjusted for multiple comparisons; Table); IL6 was below limit of detection at all timepoints, even in pts with fevers. T-cell receptor (TCR) clonotypes present in the OBX-115 infusion product were enriched in post-infusion PB and tumor (Table). Conclusions: These data support the proposed OBX-115 mechanism of action, demonstrating ACZ-driven OBX-115 TIL expansion, engraftment, and persistence; endogenous NK cell expansion, presumably driven by transactivation via mbIL15 on OBX-115, without systemic cytokine elevation; and TCR repertoire remodeling with tumor-derived, antigen-specific T cells. Investigation of OBX-115 TIL cell therapy in pts with advanced solid tumors (NCT06060613) is ongoing. Clinical trial information: NCT05470283 . Pre- and post-infusion immune profile. Characteristic, median (N=8) Baseline* D14 D28 D42 CD3-CD56+ (of live, PB), † % 14.7 27.0 35.5 56.7 CD3+ (of live, PB), † % 50.7 69.9 49.5 54.2 CD8+ (of CD3+, PB), † % 27.9 75.2 85.6 87.6 CD4+ (of CD3+, PB), † % 59.7 5.7 10.6 9.3 Ki67+ (of CD8+, PB), † % 1.5 11.1 3.7 3.2 IL15, serum, pg/mL 8.5 9.5 10.0 10.8 IL7, serum, pg/mL 2.4 3.3 3.3 2.8 OBX-115 TCR clonotypes in PB, % 14.5 80.9 69.3 59.6 OBX-115 TCR clonotypes in tumor, % 28.2 Not available 86.2 ‡ 70.4 *Pre-lymphodepletion. † n<8. ‡ D21.

Abstract Generating stem-like memory T cells (TSCM) is a potential strategy to improve adoptive immunotherapy. Elucidating optimal ways to modulate signaling pathways that enrich TSCM properties could identify approaches to achieve this goal. We discovered herein that blocking the PI3Kδ pathway pharmaceutically to varying degrees can generate T cells with increasingly heightened stemness properties, based on the progressive enrichment of the transcription factors Tcf1 and Lef1. T cells with enhanced stemness features exhibited metabolic plasticity, marked by improved mitochondrial function and glucose uptake after tumor recognition. Conversely, T cells with low or medium stemness were less metabolically dynamic, vulnerable to antigen-induced cell death, and expressed more inhibitory checkpoint receptors. Only T-cell receptor–specific or chimeric antigen receptor (CAR)-specific T cells with high stemness persisted in vivo and mounted protective immunity to tumors. Likewise, the strongest level of PI3Kδ blockade in vitro generated human tumor-infiltrating lymphocytes and CAR T cells with elevated stemness properties, in turn bolstering their capacity to regress human solid tumors. The stemness level of T cells in vitro was important, ultimately impacting their efficacy in mice bearing three distinct solid tumors. Lef1 and Tcf1 sustained antitumor protection by donor high CD8+ TSCM or CD4+ Th17SCM, as deletion of either one compromised the therapeutic efficacy. Collectively, these findings highlight the importance of strategic modulation of PI3Kδ signaling in T cells to induce stemness and lasting protective responses to solid tumors. Significance: Elevating T-cell stemness by progressively blocking PI3Kδ signaling during ex vivo manufacturing of adoptive cell therapies alters metabolic and functional properties to enhance antitumor immunity dependent on Tcf1 and Lef1.

T-cell immunoglobulin and mucin domain 3 (TIM3) is emerging as a potential target for antibody-based checkpoint blockade. However, the efficacy of TIM3 blockade in combination with other treatment modalities, has not been extensively studied. In the current work we combined TIM3 blockade with myxoma virus-based oncolytic virotherapy (OV). Our results demonstrate that myxoma virus's ability to initiate an immense antitumor immune response complements the ability of TIM3 blockade to shift the tumor microenvironment to a more proinflammatory state. As a result, the combination of TIM3 blockade and OV is able to completely eradicate established disease, while neither monotherapy is effective. These data represent the first demonstration that OV can enhance the efficacy of TIM3 blockade and suggest that this treatment may need to be incorporated into more aggressive, combinatorial regimens in order to fulfill its potential as an immunotherapeutic.

1. Mutation burden and survival using updated mutation calling system. 2. Similarity scores between samples and viral or non-viral epitopes. 3. Deconvolution of immune infiltrates. 4. Gene set enrichment analysis.

Supplementary Figure Legends for S1-4

Generating stem memory T cells (T SCM ) is a key goal for improving cancer immunotherapy. Yet, the optimal way to modulate signaling pathways that enrich T SCM properties remains elusive. Here, we discovered that the degree to which the PI3Kδ pathway is blocked pharmaceutically can generate T cells with differential levels of stemness properties. This observation was based on the progressive enrichment of transcriptional factors of stemness (Tcf-1 and Lef-1). Additional investigation revealed that T cells with high stemness features had enhanced metabolic plasticity, marked by heightened mitochondrial function and glucose uptake. Conversely, T cells with low or medium features of stemness expressed more inhibitory checkpoint receptors (Tim-3, CD39) and were vulnerable to antigen-induced cell death. Only TCR-antigen specific T cells with high stemness persisted following adoptive transfer in vivo and mounted protective immunity to melanoma tumors. Likewise, the strongest level of PI3Kδ blockade in vitro generated human tumor infiltrating lymphocytes (TILs) and CAR T cells with heightened stemness properties, in turn bolstering their capacity to regress human mesothelioma tumors. We find that the level of stemness T cells possess in vitro differentially impacts their potency upon transfer in three tumor models. Mechanistically, both Lef-1 and Tcf-1 sustain anti-tumor protection by high T SCM , as deletion of either one compromised cellular therapy. Collectively, these findings highlight the therapeutic potential of carefully modulating PI3Kδ signaling in T cells to confer high stemness and mediate protective responses to solid tumors.

We evaluated the utility of single-cell sequencing of tumor-infiltrating lymphocytes (TIL) for tumor-reactive T-cell receptor (TCR) discovery. Using the MC38 cell line as our tumor model in mice, we show that expression of exogenous TCRs via mRNA electroporation in human T cells provides an easy and quick path to validating tumor-specific candidate TCRs. We detail the identification and validation of four novel MC38-reactive mouse TCRs with varying levels of reactivity to the target cells. Validating our process, one of the MC38 TCRs is specific against a previously reported neoantigen (ASMTNMELM in the Adpgk gene). Consideration of these methodologies may aid in the development of rapid TCR-based therapies for the treatment of cancer and human disease. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

AbstractPathway Commons (https://www.pathwaycommons.org) is an integrated resource of publicly available information about biological pathways including biochemical reactions, assembly of biomolecular complexes, transport and catalysis events and physical interactions involving proteins, DNA, RNA, and small molecules (e.g. metabolites and drug compounds). Data is collected from multiple providers in standard formats, including the Biological Pathway Exchange (BioPAX) language and the Proteomics Standards Initiative Molecular Interactions format, and then integrated. Pathway Commons provides biologists with (i) tools to search this comprehensive resource, (ii) a download site offering integrated bulk sets of pathway data (e.g. tables of interactions and gene sets), (iii) reusable software libraries for working with pathway information in several programming languages (Java, R, Python and Javascript) and (iv) a web service for programmatically querying the entire dataset. Visualization of pathways is supported using the Systems Biological Graphical Notation (SBGN). Pathway Commons currently contains data from 22 databases with 4794 detailed human biochemical processes (i.e. pathways) and ∼2.3 million interactions. To enhance the usability of this large resource for end-users, we develop and maintain interactive web applications and training materials that enable pathway exploration and advanced analysis.

BACKGROUND:Oncolytic therapy uses live-replicating viruses to improve the immunological status of treated tumors. Critically, while these viruses are known to self-amplify in vivo, clinical oncolytic therapies still appear to display a strong dose dependence and the mechanisms mediating this dose dependence are not well understood. METHODS:To explore this apparent contradiction, we investigated how the initial dose of oncolytic myxoma virus affected the subsequent ability of treatment to alter the immunological status of tumors as well as synergize with programmed cell death protein 1 (PD1) blockade. RESULTS:Our results indicate that, due to viral self-amplification in vivo, the overall load of myxoma virus rapidly normalizes within treated tumors despite up to 3-log differences in inoculating dose. Because of this, therapeutic efficacy in the absence of checkpoint blockade is largely dose independent. Despite this rapid normalization, however, treatment with high or low doses of myxoma virus induces distinct immunological changes within treated tumors. Critically, these changes appear to be durably programmed based on the initial oncolytic dose with low-dose treatment failing to induce immunological improvements despite rapidly achieving equivalent viral burdens. Finally, due to the distinct immunological profiles induced by high and low myxoma virus doses, oncolytic efficacy resulting from combination with PD1 blockade therapy displays a strong dose dependence. CONCLUSIONS:Taken together, these data suggest that the ability of oncolytic myxoma virus to immunologically reprogram treated tumors is dependent on initial viral dose. Additionally, this work could provide a possible mechanistic explanation for clinical results observed with other oncolytic viruses.

This protocol repurposes Promega's T Cell Activiation Bioassay workflow to be able to test relative mouse TCR reactivity against a cell line. This specific protocol uses MC38 as the target as it doesn't normally present SIINFEKL and have good H2Kb and H2Db expression levels. The reactivity will be in relative to the positive control (OT-I reactivity against SIINFEKL-pulsed cells) and the negative control (OT-I reactiviy against unpulsed cells).

Abstract Antibodies targeting CTLA-4 induce durable responses in some patients with melanoma and are being tested in a variety of human cancers. However, these therapies are ineffective for a majority of patients across tumor types. Further understanding the immune alterations induced by these therapies may enable the development of novel strategies to enhance tumor control and biomarkers to identify patients most likely to respond. In several murine models, including colon26, MC38, CT26, and B16 tumors cotreated with GVAX, anti–CTLA-4 efficacy depends on interactions between the Fc region of CTLA-4 antibodies and Fc receptors (FcR). Anti–CTLA-4 binding to FcRs has been linked to depletion of intratumoral T regulatory cells (Treg). In agreement with previous studies, we found that Tregs infiltrating CT26, B16-F1, and autochthonous BrafV600EPten−/− melanoma tumors had higher expression of surface CTLA-4 (sCTLA-4) than other T-cell subsets, and anti–CTLA-4 treatment led to FcR-dependent depletion of Tregs infiltrating CT26 tumors. This Treg depletion coincided with activation and degranulation of intratumoral natural killer cells. Similarly, in non–small cell lung cancer (NSCLC) and melanoma patient-derived tumor tissue, Tregs had higher sCTLA-4 expression than other intratumoral T-cell subsets, and Tregs infiltrating NSCLC expressed more sCTLA-4 than circulating Tregs. Patients with cutaneous melanoma who benefited from ipilimumab, a mAb targeting CTLA-4, had higher intratumoral CD56 expression, compared with patients who received little to no benefit from this therapy. Furthermore, using the murine CT26 model we found that combination therapy with anti–CTLA-4 plus IL15/IL15Rα complexes enhanced tumor control compared with either monotherapy.

ABSTRACTPathway Commons (https://www.pathwaycommons.org) is an integrated resource of publicly available information about biological pathways including biochemical reactions, assembly of biomolecular complexes, transport and catalysis events and physical interactions involving proteins, DNA, RNA, and small molecules (e.g., metabolites and drug compounds). Data is collected from multiple providers in standard formats, including the Biological Pathway Exchange (BioPAX) language and the Proteomics Standards Initiative Molecular Interactions format, and then integrated. Pathway Commons provides biologists with (1) tools to search this comprehensive resource, (2) a download site offering integrated bulk sets of pathway data (e.g., tables of interactions and gene sets), (3) reusable software libraries for working with pathway information in several programming languages (Java, R, Python, and Javascript), and (4) a web service for programmatically querying the entire dataset. Visualization of pathways is supported using the Systems Biological Graphical Notation (SBGN). Pathway Commons currently contains data from 22 databases with 4,794 detailed human biochemical processes (i.e., pathways) and ∼2.3 million interactions. To enhance the usability of this large resource for end-users, we develop and maintain interactive web applications and training materials that enable pathway exploration and advanced analysis.

This protocol describes an imaged-based 3-dimensional (3D) cell culture cytotoxicity assay using multicellular tumor spheroids (in this case MC38 cells) sensitized with hgp100 peptide and killing them with pmel-1 T cells or the apoptosis-inducing antibiotic, Staurosporine. Our goal was to explore an in vitro 3D cell culture system to study T cell-mediated cytotoxicity as a potential way to more rapidly and relevantly test the effect of T cell manipulation on T cell cytotoxicity before moving to the mouse model. In this setup, the T cells are required to migrate towards the tumor spheroids that are already suspended in a gel matrix, there is minimal manipulation of the samples post-coculture because the samples are imaged directly in the chips to measure cell death, and there is somewhat higher throughput than confocal imaging provides (more spheroids being imaged per sample via widefield microscopy). We used 3D cell culture chips from AIM Biotech (Singapore) and adapted protocols published by: AIM Biotech: https://www.aimbiotech.com/general-protocols.html Pavesi, Andrea, Anthony T. Tan, Sarene Koh, Adeline Chia, Marta Colombo, Emanuele Antonecchia, Carlo Miccolis, et al. 2017. “A 3D Microfluidic Model for Preclinical Evaluation of TCR-Engineered T Cells against Solid Tumors.” JCI Insight 2 (12). https://doi.org/10.1172/jci.insight.89762. Jenkins, Russell W., Amir R. Aref, Patrick H. Lizotte, Elena Ivanova, Susanna Stinson, Chensheng W. Zhou, Michaela Bowden, et al. 2018. “Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids.” Cancer Discovery 8 (2): 196–215. For image analysis, refer to: Czech, Eric, Bulent Arman Aksoy, Pinar Aksoy, and Jeff Hammerbacher. 2019. “Cytokit: A Single-Cell Analysis Toolkit for High Dimensional Fluorescent Microscopy Imaging.” BMC Bioinformatics 20 (1): 448.

In vitro co-cultures of cytotoxic T cells with their target cells are important assays to asses the functionality of the T cells in a scalable way. These assays rely on co-culturing CD8 T-cells, often times genetically modified to express a specific TCR or CAR, with another type of cell line that can be recognized by T cells. Co-cultures are typically run for 6-24 hours and then the amount of cells that were killed in the co-culture can be assesed through different techniques -- e.g. radioactive Cr or non-radioactive LDH release assays. Here, we outline another alternative to these release assays which relies on flow cytometry to estimate the number of target cells left in the culture after a certain period of time.

Three-dimensional (3D) cell culture systems with tumor spheroids are being adopted for research on the antitumor activity of drug treatments and cytotoxic T cells. Analysis of the cytotoxic effect on 3D tumor cultures within a 3D scaffold, such as collagen, is challenging. Image-based approaches often use confocal microscopy, which greatly limits the sample size of tumor spheroids that can be assayed. We explored a system where tumor spheroids growing in a collagen gel within a microfluidics chip can be treated with drugs or co-cultured with T cells. We attempted to adapt the system to measure the death of cells in the tumor spheroids directly in the microfluidics chip via automated widefield fluorescence microscopy. We were able to successfully measure drug-induced cytotoxicity in tumor spheroids, but had difficulties extending the system to measure T cell-mediated tumor killing.