Tumor-infiltrating T Lymphocytes Research Articles

Abstract 3740: Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor

Abstract Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC) targeted to carcino-embryonic antigen-related cell adhesion molecule 5 (CEACAM5) and delivering DM4, a maytansinoid cytotoxic. CEACAM5 is highly expressed in several tumor types, including non-squamous non-small cell lung cancer (NSQ NSCLC). It is currently being evaluated in patients with advanced CEACAM5-positive NSQ NSCLC, in a phase II study in combination with pembrolizumab, and a phase III trial as single agent in comparison with docetaxel. Preclinical investigations were performed to determine whether SAR408701 could be beneficially combined with immune checkpoint inhibitors. We, first, demonstrated that the tusamitamab ravtansine payload, DM4, is able to induce robust immunogenic cell death (ICD) using an in vivo vaccination assay. In this assay, immunocompetent mice were “vaccinated” by DM4-treated CT26 cells and then later rechallenged with CT26 tumor cells. Tumor rejection was observed in more than 50% of “vaccinated” mice. The ICD was characterized by the expression or release of danger-associated molecular patterns (DAMPs), and we observed DAMP modulation such as calreticulin (CRT) exposure at the cell surface of dying tumor cells, and the release of the High-Mobility Group Box 1 (HMGB1) in the extracellular space by CT26 cells in response to DM4 treatment. We also showed that DM4-treated CT26 tumor cells implanted into mice or SAR408701 treatment of MC38 tumor-bearing mice induced an elevation of plasmatic level of murine IL-6 and INFγ, revealing potential antitumor immunity modulation by the ADC and its payload. Immunohistochemistry analysis of MC38 tumors also revealed that tusamitamab ravtansine treatment induced a dose-dependent increase of CD8 T-cell infiltration in tumors correlating to dose-dependent efficacy. Finally, tusamitamab ravtansine was combined with immune checkpoint inhibitors (ICIs) in immunocompetent mice bearing MC38 tumor model. In this model, the combination of tusamitamab ravtansine with anti-PD-1 or anti-PD-L1 antibody led to complete response and, even, tumor-free survivors at 120 days post tumor implantation while single agents were inactive or transiently active. In summary, these preclinical data show that tusamitamab ravtansine could be beneficially combined with immune checkpoint and strongly support their evaluation in clinical development. Citation Format: Celine Nicolazzi, Anne-Marie Lefebvre, Nicolas Moindrot, Christelle Larois, Marion Classe, Sukhvinder Sidhu. Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor [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 3740.

Abstract 3961: Impact of CD8 resident memory T cells on antitumor immunity and response to cancer immunotherapy

Abstract CD8+ resident memory T (TRM) cells expressing the CD103 integrin accumulate in human lung tumors and are associated with a favorable prognosis. We have previously demonstrated that a high density of CD103+CD8+ T cells in human lung tumors is a biomarker of response to immune checkpoint blockade (ICB) immunotherapy. However, a large fraction of human tumors is only weakly infiltrated by CD8+ TRM cells, and the reason of this TRM desert is not explained. Therefore, defining the molecular signals that give rise and maintain TRM cells within the tumor microenvironment (TME) and the mechanisms that potentiate antitumor TRM functions are important challenges. Data obtained in pre-clinical mouse colorectal cancer and melanoma models demonstrate that knocking-out CD103 impairs antitumor T-cell immunity and response to ICB and therapeutic peptide vaccine. Moreover, immunization of melanoma-bearing mice with the cancer vaccine results in a dramatic decrease in the percentage of CD103+ TRM cells among CD8+ tumor-infiltrating T lymphocytes (TIL). Algorithm analyses applied to spectral cytometry data of TIL support the conclusion that upon activation with the peptide vaccine a TRM subset gives rise to tumor-specific effector T cells. The mechanisms associated with the decrease in CD103 expression on CD8+ T cells and lead to a TRM differentiation shift will be presented as well as the key role of TGF-b in CD8+CD103+ TRM formation and persistence in the TME. Our data demonstrate that tumor CD8+CD103+ TRM are capable of mounting a potent antitumor immunity and suggest that a fine balance between diverse signals in the tumor ecosystem is required to induce their differentiation and thereby optimize response to cancer immunotherapy. Citation Format: Stephanie Corgnac, Isabelle DAMEI, Fathia Mami-Chouaib. Impact of CD8 resident memory T cells on antitumor immunity and response to cancer immunotherapy [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 3961.

Abstract 4057: Beyond IL-2: The promise of anti PD-1-attenuated IL-2 fusion protein in cancer immunotherapy

Abstract PD1+ CD8+ tumor-infiltrating T-cells have been at the forefront of cancer immunotherapies due to their antigen-specificity, cytotoxicity, proliferative capacity, and stem-like/memory properties. IL-2 is essential for T cell growth and proliferation, however, its systemic administration at a high dose was associated with intolerable toxicities, while low-dose treatment contradicted the intended therapeutic effect by activating regulatory T cells.To overcome the limitations of IL-2, we fused modified IL-2 sequences with reduced affinity for the murine IL-2 receptor to a PD1 antibody designed to target the PD1+ T cells found in tumors. The resulting murine PD1 antibody-attenuated-IL-2 (mAnti-PD1-IL2Att) demonstrated similar IL-2 potency on PD1+ cells as native IL-2, but diminished potency by orders of magnitude on PD1- cells. The agent was furthermore designed to bind PD1 non-competitively with known PD1-blocking antibodies, so that it could be used in combination with them to achieve effective anti-tumor response. We demonstrated that mAnti-PD1-IL2Att exhibits potent anti-tumor activity, resulting in complete tumor regression in a syngeneic MC38 colorectal cancer model, and significant anti-tumor activity in other models of different tumor types. The mechanism was evaluated by immunophenotyping and single-cell RNAseq. Shortly after the first administration of mAnti-PD1-IL2Att, CD8+ T cells, rather than Tregs, emerged as the primary responding cells; these CD8+ T cells exhibited increased proliferation and activation with reduced expression of exhaustion markers (PD1+Tim3+). Following subsequent doses, there was a notable increase in the number of effector cells, followed by a prolonged generation of effector and memory cells at a later stage. Myeloid cells exhibited increased transcriptional programs related to chemotaxis, co-stimulation, and co-inhibition, including the upregulation of the PD1-PDL1 pathway. By contrast, blockade of PD1 with a naked PD1 antibody led to distinct cellular responses and less pronounced anti-tumor activity, and a combination of the blocking antibody with PD1-attIL-2 was superior to either agent alone. Together, our findings suggest that mAnti-PD1-IL2Att potent anti-tumor activity was driven by its direct targeting of PD1+ on tumor-infiltrating CD8+ T-cells, shedding light on the specific changes in their dynamic differentiation trajectory and their impact on the tumor microenvironment. Citation Format: Sarah Amar, Yulia Liubomirski, Eilam Yeini, Michael Monsonego, Tomer Weiss, Galia Tiram, Anshika Katyal, Opal Avramoff, Anne Krinsky, Keren Reshef, Shai Dulberg, Ayelet Kaminitz, Dana Bar On, Inbal Ben Eliezer, Ortal Iancu, Tetsuya Taura, David Wilson, Paul Ayton, Ronit Satchi-Fainaro, Asaf Madi. Beyond IL-2: The promise of anti PD-1-attenuated IL-2 fusion protein in cancer immunotherapy [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 4057.

Abstract 6869: Paired single-cell B-cell transcriptomics and receptor sequencing reveal activation states and clonal signatures that characterize B cell immunity in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is the second most common adult leukemia with a five-year survival rate around 30%. For decades, AML treatment has centered around intensive chemotherapy frequently followed by allogeneic stem cell transplant (Allo-SCT). The power of Allo-SCT to induce lasting remission has been attributed to a potent graft-versus-leukemia effect and has inspired efforts to stimulate host immunity against AML via immune checkpoint blockade (ICB). Thus far, these studies have yielded mixed results. While tumor-infiltrating T-cells have long been the focus of immunotherapy investigations, a growing body of data points to a central role of B-cells in mediating anti-tumor immunity, and a comprehensive assessment of B lymphocytes within the AML immune microenvironment has never been carried out with single cell clonal resolution. We performed paired single-cell transcriptomic and B cell receptor (BCR) analysis on 52 bone marrow aspirate samples. These samples included 6 from healthy bone marrow donors (normal), 24 from newly diagnosed AML patients (NewlyDx), and 22 from 8 relapsed or refractory AML patients (RelRef), who underwent assessment both before and after ICB with azacitidine/nivolumab. We first used transcriptomic data to delineate canonical B-cell subpopulations and observed a reduction in nascent B cells and an expansion of differentiated B cells in AML patients (RelRef>NewlyDx). Overlaying BCR sequencing and clonotypic analysis revealed clonal expansion, low clonotypic diversity, and extensive somatic hypermutation in the RelRef samples, suggesting antigen-driven affinity maturation within the tumor microenvironment. Furthermore, we identified robust AP-1 expression (a marker of activation) in B-cells from NewlyDx patients and a marked loss of AP-1 in RelRef patients, suggesting that B-cell exhaustion may be a key immunologic feature of AML relapse. Importantly, we observed that AP-1 activity is restored and that specific B-cell clones expand after ICB, but only among patients who clinically respond to ICB. In line with these observations, we also noted the clonal expansion of AML-associated plasma cells and loss of clonotypic diversity among ICB clinical responders, pointing to an antitumor role for these clones. Finally, we characterized atypical memory B cells which demonstrate high antigen presentation activity, close interaction with AML cells and are associated with unfavorable clinical outcomes, suggesting that they play a direct role in aiding AML survival and immune escape. Collectively, our findings establish the dynamic landscape of AML-associated B-cells while identifying specific B-cell subpopulations and B-cell-AML interactions that may be targeted by the next generation of AML-directed immunotherapies. Citation Format: Shengnan Guo, Gopi S. Mohan, Bofei Wang, Tianhao Li, Naval Daver, Yuting Zhao, Patrick K. Reville, Dapeng Hao, Hussein A. Abbas. Paired single-cell B-cell transcriptomics and receptor sequencing reveal activation states and clonal signatures that characterize B cell immunity in acute myeloid leukemia [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 6869.

Abstract 1594: Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response

Abstract Background: While T-cell-based immunotherapy emerged as a promising tool for cancer management, clinical trials and translational studies revealed that cancer cells develop resistance by modulating tumor microenvironment and cell-intrinsic mechanisms. Pancreatic cancer (PC), consisting of dense stroma, immunosuppressive environment, and aberrant mucin expression, has a dismal survival rate and responds poorly to immunotherapies. MUC4, a member of the mucin family, has been reported to block lymphokine-activated killer cells and induce apoptosis of cytotoxic T-cells, suggesting its possible role in immune modulation. However, how the T-cell secretome influences MUC4 expression in PC and its role in modulating the T-cell response is poorly investigated. Method: The scRNA-seq data analysis and primary T-cell conditioned media (CM) were utilized to investigate the MUC4 and T-cell crosstalk. T-cell CM-treated murine PC cell line (KCT-3266) was analyzed by RNA-seq analysis followed by MUC4 silencing studies to investigate its role in evading T-cell response. The subcutaneous murine model was utilized to investigate the influence of MUC4 KO on immune cells and cytotoxic cell infiltration. The RNA isolated from tumor tissues from KrasG12D/+, Trp53R127H/+, Pdx-1-Cre (KPC), and Muc4 knockout (KPMC) murine models were used for PanCancer immune profiling. Results: The scRNA-seq analysis suggests that intratumoral T-cells positively correlate with MUC4 expression in PC patients. The activated T-cell CM consisting of predominantly IL-2, IFN-γ, and TNF-α cytokines, significantly induce the expression of MUC4 transcriptionally and translationally in both human (SW1990 and COLO357) and murine (KCT-3248 and KCT3266) PC cell lines. RNA-seq analysis from T-cell CM-treated cancer cells revealed that T-cell secretome induced the pathways related to cancer cell death. The CRISPR knockout of MUC4 in PC cells showed increased expression of cleaved caspase-3 after T-cell CM treatment, indicating that MUC4 plays a protective role against the T-cell CM-mediated killing. Subcutaneous implantation of MUC4 proficient and deficient PC cells on the flanks of C57BL/6 immunocompetent mice demonstrated a significantly higher infiltration of CD3 positive and cytotoxic T-cell infiltration in the MUC4 deficient tumors, resulting in significantly lower tumor weight. The PanCancer immune profiling also showed that depletion of Muc4 increases the T-cells and cytotoxic T-cell signaling scores in the KPMC tumor tissues compared to KPC. Conclusion: T-cell secretome induces MUC4 expression in pancreatic cancer cells, and the increased MUC4 expression reduces T-cell infiltration and protects PC cells against T-cell-mediated killing. Citation Format: Xiaoqi Li, Imran Khan, Rachel Kehrberg, Zahraa Wajih Alsafwani, Rakesh Bhatia, Sushil Kumar, Surinder K. Batra. Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response [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 1594.

Abstract 7048: Multiomics characterization of breast angiosarcoma from an Asian cohort reveals enrichment for angiogenesis signaling pathway and tumor-infiltrating macrophages

Abstract Recent epidemiological data suggests a rising incidence of breast angiosarcoma (AS-B) in the Western population, with over two-thirds related to irradiation or chronic lymphedema. However, unlike head and neck angiosarcoma (AS-HN), AS-B disease characteristics in Asia remains unclear. We examined clinical patterns of angiosarcoma patients (n = 176) seen in an Asian tertiary cancer centre from 1999 to 2021, and specifically investigated the molecular and immune features of AS-B in comparison to AS-HN. Data from whole genome sequencing (WGS) (AS-HN, n = 44; breast, n = 7), NanoString PanCancer IO360 gene expression profiling (AS-HN, n = 42; breast, n = 7) and 10X Visium spatial transcriptomics (AS-HN, n = 4; breast, n = 2) were analyzed. Majority of cases were AS-HN (n = 104; 59.1%), while AS-B (n = 16, all females) accounted for 9.1% of the cases. The median age at diagnosis was 43 years (range, 26 to 74). Four cases (25%) were related to prior irradiation, while 6 out of 8 cases evaluated were positive for human herpesvirus-7 on immunohistochemistry. Although all cases were non-metastatic at diagnosis and were treated with curative-intent surgery, 6 patients (37.5%) experienced rapid relapse within a median of 7.8 months. Based on WGS, 4 of the 7 AS-B had non-synonymous somatic variants in 48 genes (range, 2 to 28 per case). Among the notable variants found were in kinases (KDR, JAK2, FLT4, ATR, EPHB1 and PRKACA). Other variants were found in cancer-related genes (e.g. ATRX, FOXO1, SMAD3, PTCH1, NOTCH4, HIF1A and SETD1A). These genes were functionally annotated by performing over representation analysis (www.webgestalt.org) on publicly available databases (e.g. Gene Ontology and KEGG). They were enriched in cancer-related pathways such as regulation of cell differentiation, VEGFR and receptor tyrosine kinase signaling pathways. By NanoString profiling, compared to AS-HN, AS-B was enriched for angiogenesis, notch signaling and metastasis-associated matrix remodeling pathways. Conversely, AS-B was depleted of lymphoid compartment, immune cell adhesion and migration expression signatures. Based on the gene expression signatures, AS-B was enriched for macrophages, CD8+ T cells and was depleted for T-reg cells. Furthermore, spatial transcriptomics showed that compared to AS-HN, tumor-infiltrating leukocytes (TILs) in AS-B were enriched for macrophage and T-cells but were depleted for B-cells and NK cells. The location of TILs could provide information on the feasibility of immunotherapy in angiosarcoma. In conclusion, we observed a convergence of both mutational and expression signatures on angiogenesis signaling pathways in our AS-B cases. The spatial transciptomic data revealed that AS-B was enriched for tumor-infiltrating macrophages and T-cells which might have implications on the role of immunotherapy. Citation Format: Tun Kiat Ko, Zexi Guo, Bavani Kannan, Boon Yee Lim, Jing Yi Lee, Abner Herbert Lim, Zhimei Li, Elizabeth Chun Yong Lee, Cedric Chuan-Young Ng, Bin Tean Teh, Jason Yongsheng Chan. Multiomics characterization of breast angiosarcoma from an Asian cohort reveals enrichment for angiogenesis signaling pathway and tumor-infiltrating macrophages [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 7048.

Abstract 70: Unlocking the potential of the T-cell receptor in prostate cancer

Abstract Introduction: Prostate cancer (PC) is the most commonly diagnosed cancer among western men, with its diagnosis and subsequent monitoring still heavily dependent on the prostate specific antigen (PSA) test. However, the prognostic accuracy remains poor, leading to overtreatment of patients with non-aggressive cancer and undertreatment of those with aggressive cancer in need of intervention. Malignant cells are subject to constant monitoring and elimination by T-cell lymphocytes recognizing cancer-specific antigens through the T-cell receptor (TCR). Consequently, to establish itself and progress, cancer must evade or modulate the surveillance mechanisms of the immune system. As cancer growth accelerates, immune evasion becomes more difficult, resulting in a more pronounced impact on the immune system. Recently, our research group documented the clinical value of tumor-infiltrating T-cells as markers of aggressive PC. Here, we hypothesize that this signal can also be found in circulating T-cells isolated from the blood and used as proxy for the antitumoral immune response in tissue. Methods: This study utilizes a diverse and well-characterized cohort, encompassing 220 castration resistant PC patients (CRPC) with blood samples taken at the time of CRPC diagnosis, 30 newly diagnosed hormone-naïve PC patients, and 30 healthy male donors. For a deeper understanding of the mechanisms underlying the antitumoral immune response and its impact on disease progression, a subset of CRPC patients (n = 25) are TCR sequenced throughout their disease course and uniquely, the TCR repertoire of their metastatic biopsies is characterized. Results: In an initial comparison of 8 CRPC patients and 8 age-matched healthy males, we observed that cancer patients had a higher proportion of hyper-expanded T-cell clones (P = 0.003; Fisher’s exact test) and a less diverse TCR repertoire in blood. Furthermore, 86.5 % of hyper-expanded clones found in peripheral blood samples could also be found in malignant prostate tissue from matched CRPC patients, while overlap between patients was virtually non-existent Conclusion: Our pilot study detected differences in the TCR repertoire between cancer patients and matched controls. The clones contributing most to this difference were concurrently found in malignant tissue indicating that the antitumoral immune response can indeed be detected in the periphery. Given these promising results, we are currently running the larger study described above to obtain a better understanding of the TCR repertoire and establish its prognostic potential in PC. While the findings showcased in this abstract exclusively stem from the pilot study, results for the full cohort will be presented at the conference. Citation Format: Eske Nøhr Glud, Jacob H. Fredsøe, Iver K. Nordentoft, Asbjørn Kjær, Maria Rusan, Britt E. Laursen, Bodil G. Pedersen, Nicolai J. Birkbark, Lars Dyrskjøt, Michael Borre, Karina Dalsgaard Sørensen. Unlocking the potential of the T-cell receptor in prostate 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 70.

Abstract 1530: Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice

Abstract The relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2−/−, IL2Rγ−/−, SIRPαNOD and Flt3−/−) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models overexpressing human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and test novel therapeutics. Effect of Flt3L-induced boost on myeloid compartment is seen in a non-small cell lung cancer (A549) tumor model. A single boost prior to tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the tumor microenvironment (TME). Human triple negative cancer cell (TNBC) MDA-MB-231 and human pancreatic adenocarcinoma HPAFII cell lines are widely used in cancer research and drug development. We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft (CDX) is CD34+ donor independent and the TME composition varies according to cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231 bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII bearing mice is mainly composed of T cells enabling T cells-based therapy. Treatment of BRGSF-HIS mice bearing HPAFII tumor cells with combotherapy based on CD3xTAA + CD28xTAA efficiently reduces tumor growth in vivo compared to vehicle-treated mice or mice injected with CD3xTAA alone. Induced systemic immunomodulation is observed in the TME where increased numbers of both CD4 and CD8 T cells are observed upon combotherapy. Remarkably, while no CD34+ donor effect is observed in CDX growth kinetic, response to treatment appears to be donor dependent with apparition of “weak” and “good” responders. This heterogeneity of response mimics what is observed in clinic and enables further investigations of treatment mode of action and immune escape mechanisms. Citation Format: Perrine Martin-Jeantet, Siham Hedir, Fabiane Sonego, Gaëlle Martin, Yacine Cherifi, Kader Thiam. Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice [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 1530.

Immunogenicity and effectiveness of an mRNA therapeutic vaccine for HPV-related malignancies.

Human papillomavirus (HPV) infections account for several human cancers. There is an urgent need to develop therapeutic vaccines for targeting preexisting high-risk HPV (such as HPV 16 and 18) infections and lesions, which are insensitive to preventative vaccines. In this study, we developed a lipid nanoparticle-formulated mRNA-based HPV therapeutic vaccine (mHTV), mHTV-02, targeting the E6/E7 of HPV16 and HPV-18. mHTV-02 dramatically induced antigen-specific cellular immune response and robust memory T-cell immunity in mice, besides significant CD8+ T-cell infiltration and cytotoxicity in TC-1 tumors expressing HPV E6/E7, resulting in tumor regression and prolonged survival in mice. Moreover, evaluation of routes of administration found that intramuscular or intratumoral injection of mHTV-02 displayed significant therapeutic effects. In contrast, intravenous delivery of the vaccine barely showed any benefit in reducing tumor size or improving animal survival. These data together support mHTV-02 as a candidate therapeutic mRNA vaccine via specific administration routes for treating malignancies caused by HPV16 or HPV18 infections.

Acetyl-CoA carboxylase obstructs CD8+ T cell lipid utilization in the tumor microenvironment

The solid tumor microenvironment (TME) imprints a compromised metabolic state in tumor-infiltrating Tcells (TILs), hallmarked by the inability to maintain effective energy synthesis for antitumor function and survival. Tcells in the TME must catabolize lipids via mitochondrial fatty acid oxidation (FAO) to supply energy in nutrient stress, and it is established that Tcells enriched in FAO are adept at cancer control. However, endogenous TILs and unmodified cellular therapy products fail to sustain bioenergetics in tumors. We reveal that the solid TME imposes perpetual acetyl-coenzyme A (CoA) carboxylase (ACC) activity, invoking lipid biogenesis and storage in TILs that opposes FAO. Using metabolic, lipidomic, and confocal imaging strategies, we find that restricting ACC rewires Tcell metabolism, enabling energy maintenance in TME stress. Limiting ACC activity potentiates a gene and phenotypic program indicative of Tcell longevity, engendering Tcells with increased survival and polyfunctionality, which sustains cancer control.

DDX3 regulates cancer immune surveillance via 3′ UTR-mediated cell-surface expression of PD-L1

Programmed death-1 (PD-1)/PD ligand-1 (PD-L1)-mediated immune escape contributes to cancer development and has been targeted as an anti-cancer strategy. Here, we show that inhibition of the RNA helicase DDX3 increased CD8+ Tcell infiltration in syngeneic oral squamous cell carcinoma tumors. DDX3 knockdown compromised interferon-γ-induced PD-L1 expression and, in particular, reduced the level of cell-surface PD-L1. DDX3 promoted surface PD-L1 expression by recruiting the adaptor protein 2 (AP2) complex to the 3' UTR of PD-L1 mRNA. DDX3 depletion or 3' UTR truncation increased the binding of the coatomer protein complexes to PD-L1, leading to its intracellular accumulation. Therefore, this 3' UTR-dependent mechanism may counteract cellular negative effects on surface trafficking of PD-L1. Finally, pharmaceutic disruption of DDX3's interaction with AP2 reduced surface PD-L1 expression, supporting that the DDX3-AP2 pathway routes PD-L1 to the cell surface. Targeting DDX3 to modulate surface trafficking of immune checkpoint proteins may provide a potential strategy for cancer immunotherapy.

Lactate modulates RNA splicing to promote CTLA-4 expression in tumor-infiltrating regulatory T cells

RNA splicing is involved in cancer initiation and progression, but how it influences host antitumor immunity in the metabolically abnormal tumor microenvironment (TME) remains unclear. Here, we demonstrate that lactate modulates Foxp3-dependent RNA splicing to maintain the phenotypic and functional status of tumor-infiltrating regulatory T (Treg) cells via CTLA-4. RNA splicing in Treg cells was correlated with the Treg cell signatures in the TME. Ubiquitin-specific peptidase 39 (USP39), a component of the RNA splicing machinery, maintained RNA-splicing-mediated CTLA-4 expression to control Treg cell function. Mechanistically, lactate promoted USP39-mediated RNA splicing to facilitate CTLA-4 expression in a Foxp3-dependent manner. Moreover, the efficiency of CTLA-4 RNA splicing was increased in tumor-infiltrating Treg cells from patients with colorectal cancer. These findings highlight the immunological relevance of RNA splicing in Treg cells and provide important insights into the environmental mechanism governing CTLA-4 expression in Treg cells.

Combination Immunotherapy of Oncolytic Flu-Vectored Virus and Programmed Cell Death 1 Blockade Enhances Antitumor Activity in Hepatocellular Carcinoma.

Oncolytic viruses (OVs) are appealing anti-tumor agents. But it is limited in its effectiveness. In this study, we used combination therapy with immune checkpoint inhibitor to enhance the antitumor efficacy of OVs. Using reverse genetics technology, we rescued an oncolytic influenza virus with the name delNS1-GM-CSF from the virus. After identifying the hemagglutination and 50% tissue culture infectivedose (TCID50) of delNS1-GM-CSF, it was purified, and the viral morphology was observed under electron microscopy. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) was used to identify the level of GM-CSF expression in delNS1-GM-CSF, and the GM-CSF expression level was determined after infection with delNS1-GM-CSF by enzyme linked immunosorbent assay (ELISA). To study the tumor-killing effect of delNS1-GM-CSF, we utilized the hepatocellular carcinoma (HCC) tumor-bearing mouse model. To examine signaling pathways, we performed transcriptome sequencing on mouse tumor tissue and applied western blotting to confirm the results. Changes in T-cell infiltration in HCC tumors following treatment were analyzed using flow cytometry and immunohistochemistry. DelNS1-GM-CSF can target and kill HCCs without damaging normal hepatocytes. DelNS1-GM-CSF combined with programmed cell death 1 blockade therapy enhanced anti-tumor effects and significantly improved mouse survival. Further, we found that combination therapy had an antitumor impact via the janus kinase-signal transducer and activator of transcription (JAK2-STAT3) pathway as well as activated CD4+ and CD8+T cells. Interestingly, combined therapy also showed promising efficacy in distant tumors. DelNS1-GM-CSF is well targeted. Mechanistic investigation revealed that it functions through the JAK2-STAT3 pathway. Combination immunotherapies expected to be a novel strategy for HCC immunotherapy.

Revolutionizing cancer treatment: the power of bi- and tri-specific T-cell engagers in oncolytic virotherapy.

Bi- or tri-specific T cell engagers (BiTE or TriTE) are recombinant bispecific proteins designed to stimulate T-cell immunity directly, bypassing antigen presentation by antigen-presenting cells (APCs). However, these molecules suffer from limitations such as short biological half-life and poor residence time in the tumor microenvironment (TME). Fortunately, these challenges can be overcome when combined with OVs. Various strategies have been developed, such as encoding secretory BiTEs within OV vectors, resulting in improved targeting and activation of T cells, secretion of key cytokines, and bystander killing of tumor cells. Additionally, oncolytic viruses armed with BiTEs have shown promising outcomes in enhancing major histocompatibility complex I antigen (MHC-I) presentation, T-cell proliferation, activation, and cytotoxicity against tumor cells. These combined approaches address tumor heterogeneity, drug delivery, and T-cell infiltration, offering a comprehensive and effective solution. This review article aims to provide a comprehensive overview of Bi- or TriTEs and OVs as promising therapeutic approaches in the field of cancer treatment. We summarize the cutting-edge advancements in oncolytic virotherapy immune-related genetic engineering, focusing on the innovative combination of BiTE or TriTE with OVs.

Abstract A020: An early determination of patients eligibility for a bladder cancer immunotherapy using a data science approach

Abstract Advanced bladder cancer patients have limited treatment options and, in most cases, limited overall survival. The first clinical trial with adoptive T cell therapy (ACT) that uses bladder cancer patients’ autologous tumor-infiltrating T lymphocytes (TILs) has been recently opened at Moffitt. To help identify which patients could benefit from ACT-TIL we are developing a machine learning predictor that takes advantage of tumor evolution data, such as tumor stages and grades routinely collected in clinic. The first phase of ACT-TIL involves resection of the primary tumor and the growth of TILs followed by a rapid expansion protocol of the reactive T cells. It may take up to 4-5 weeks before bladder cancer patients can get their own expanded TILs. Our predictor aims to determine whether the tumor isolated from a patient will grow TILs. In this work, we present a machine learning predictor of expansion of tumor infiltrating lymphocytes (ML-PETIL) that uses retrospectively collected patient tumor data with either demographic, clinical, or biological sample-related features. The proposed method consists of a suite of algorithms to identify a robust set of predictive features (feature selection algorithms) that are used to discriminate the dataset into two classes-that with successful TIL expansion or with no TIL growth (classification algorithms). Our predictor includes metrics which are used to address data imbalance and handle small to medium-sized datasets that contain features of varying datatypes and magnitudes. The ML-PETIL protocol offers an early determination of patients who would be eligible for ACT-TIL therapy. Citation Format: Kayode D. Olumoyin, Ahmet M. Aydin, Brittany L Bunch, Shari Pilon-Thomas, Michael Poch, Katarzyna A. Rejniak. An early determination of patients eligibility for a bladder cancer immunotherapy using a data science approach [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A020.

Benzosceptrin C induces lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting DHHC3

Programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blockade has become a mainstay of cancer immunotherapy. Targeting the PD-1/PD-L1 axis with small molecules is an attractive approach to enhance antitumor immunity. Here, we identified a natural marine product, benzosceptrin C (BC), that enhances the cytotoxicity of Tcells to cancer cells by reducing the abundance of PD-L1. Furthermore, BC exerts its antitumor effect in mice bearing MC38 tumors by activating tumor-infiltrating Tcell immunity. Mechanistic studies suggest that BC can prevent palmitoylation of PD-L1 by inhibiting DHHC3 enzymatic activity. Subsequently, PD-L1 is transferred from the membrane to the cytoplasm and cannot return to the membrane via recycling endosomes, triggering lysosome-mediated degradation of PD-L1. Moreover, the combination of BC and anti-CTLA4 effectively enhances antitumor Tcell immunity. Our findings reveal a previously unrecognized antitumor mechanism of BC and represent an alternative immune checkpoint blockade (ICB) therapeutic strategy to enhance the efficacy of cancer immunotherapy.

Tumor-infiltrating T lymphocytes evaluated using digital image analysis predict the prognosis of patients with diffuse large B-cell lymphoma.

The implication of the presence of tumor-infiltrating T lymphocytes (TIL-T) in diffuse large B-cell lymphoma (DLBCL) is yet to be elucidated. We aimed to investigate the effect of TIL-T levels on the prognosis of patients with DLBCL. Ninety-six patients with DLBCL were enrolled in the study. The TIL-T ratio was measured using QuPath, a digital pathology software package. The TIL-T ratio was investigated in three foci (highest, intermediate, and lowest) for each case, resulting in TIL-T-Max, TIL-T-Intermediate, and TIL-T-Min. The relationship between the TIL-T ratios and prognosis was investigated. When 19% was used as the cutoff value for TIL-T-Max, 72 (75.0%) and 24 (25.0%) patients had high and low TIL-T-Max, respectively. A high TIL-T-Max was significantly associated with lower serum lactate dehydrogenase levels (p < .001), with patient group who achieved complete remission after RCHOP therapy (p < .001), and a low-risk revised International Prognostic Index score (p < .001). Univariate analysis showed that patients with a low TIL-T-Max had a significantly worse prognosis in overall survival compared to those with a high TIL-T-Max (p < .001); this difference remained significant in a multivariate analysis with Cox proportional hazards (hazard ratio, 7.55; 95% confidence interval, 2.54 to 22.42; p < .001). Patients with DLBCL with a high TIL-T-Max showed significantly better prognosis than those with a low TIL-T-Max, and the TIL-T-Max was an independent indicator of overall survival. These results suggest that evaluating TIL-T ratios using a digital pathology system is useful in predicting the prognosis of patients with DLBCL.

Characterization of natural killer and cytotoxic T-cell immune infiltrates in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor response to systemic therapies, including immunotherapy. Given the immunotherapeutic potential of natural killer (NK) cells, we evaluated intratumoral NK cell infiltrates along with cytotoxic T cells in PDAC to determine their association with patient outcomes. We analyzed tumors from 93 PDAC patients treated from 2012 to 2020. Predictor variables included tumor-infiltrating lymphocytes (TILs), T-cell markers (CD3, CD8, CD45RO), NK marker (NKp46), and NK inhibitory marker (major histocompatibility complexclass I[MHC-I]) by immunohistochemistry. Primary outcome variables were recurrence-free survival (RFS) and overall survival (OS). Mean TILs, CD3, and NKp46 scores were 1.3 ± 0.63, 20.6 ± 17.5, and 3.1 ± 3.9, respectively. Higher expression of CD3 and CD8 was associated with higher OS, whereas NK cell infiltration was not associated with either RFS or OS. There was a tight positive correlation between MHC-I expression and all T-cell markers, but not with NKp46. Overall NK cell infiltrates were low in PDAC and did not predict clinical outcomes, whereas T-cell infiltrates did. Further characterization of the immune infiltrate in PDAC, including inhibitory signals and suppressive cell types, may yield better biomarkers of prognosis and immune targeting in this refractory disease.

Characterization of immunomodulating agents from Staphylococcus aureus for priming immunotherapy in triple-negative breast cancers

Immunotherapy, specifically immune checkpoint blockade (ICB), has revolutionized the treatment paradigm of triple-negative breast cancers (TNBCs). However, a subset of TNBCs devoid of tumor-infiltrating T cells (TILs) or PD-L1 expression generally has a poor response to immunotherapy. In this study, we aimed to sensitize TNBCs to ICB by harnessing the immunomodulating potential of S. aureus, a breast-resident bacterium. We show that intratumoral injection of spent culture media from S. aureus recruits TILs and suppresses tumor growth in a preclinical TNBC model. We further demonstrate that α-hemolysin (HLA), an S. aureus-produced molecule, increases the levels of CD8+ T cells and PD-L1 expression in tumors, delays tumor growth, and triggers tumor necrosis. Mechanistically, while tumor cells treated with HLA display Gasdermin E (GSDME) cleavage and a cellular phenotype resembling pyroptosis, splenic T cells incubated with HLA lead to selective expansion of CD8+ T cells. Notably, intratumoral HLA injection prior to ICB augments the therapeutic efficacy compared to ICB alone. This study uncovers novel immunomodulatory properties of HLA and suggests that intratumoral administration of HLA could be a potential priming strategy to expand the population of TNBC patients who may respond to ICB.

T-Cell-Based Platform for Functional Screening of T-Cell Receptors Identified in Single-Cell RNA Sequencing Data Sets of Tumor-Infiltrating T-Cells.

The advent of single-cell RNA sequencing (scRNAseq) has enabled in-depth gene expression analysis of several thousand cells isolated from tissues. We recently reported the application of scRNAseq toward the dissection of the tumor-infiltrating T-cell repertoire in human pancreatic cancer samples. In this study, we demonstrated that combined whole transcriptome and T-cell receptor (TCR) sequencing provides an effective way to identify tumor-reactive TCR clonotypes on the basis of gene expression signatures. An important aspect in this respect was the experimental validation of TCR-mediated anti-tumor reactivity by means of an in vitro functional assay, which is the subject of the present protocol. This assay involves the transient transfection of mRNA gene constructs encoding TCRα/β pairs into a well-defined human T-cell line, followed by co-cultivation with the tumor cells of interest and detection of T-cell activation by flow cytometry. Due to the high transfectability and the low background reactivity of the mock-transfected T-cell line to a wide variety of tumor cells, this assay offers a highly robust and versatile platform for the functional screening of large numbers of TCR clonotypes as identified in scRNAseq data sets. Whereas the assay was initially developed to test TCRs of human origin, it was more recently also applied successfully for the screening of TCRs of murine origin. Key features • Efficient functional screening of-and discrimination between-TCRs isolated from tumor-reactive vs. bystander T-cell clones. • Applicable to TCRs from CD8+ and CD4+ tumor-infiltrating T-cells originating from patient-derived tumor samples and syngeneic mouse tumor models. • Rapid flow cytometric detection of T-cell activation by means of TNFα and CD107a expression after a 5 h T-cell/tumor cell co-cultivation.