Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy

Antibodies targeting costimulatory receptors of T cells have been developed for the activation of T cell immunity in cancer immunotherapy. However, costimulatory molecule expression is often lacking in tumor-infiltrating immune cells, which can impede antibody-mediated immunotherapy. Here, we hypothesize that delivery of costimulatory receptor mRNA to tumor-infiltrating T cells will enhance the antitumor effects of antibodies. We first design a library of biomimetic nanoparticles and find that phospholipid nanoparticles (PL1) effectively deliver costimulatory receptor mRNA (CD137 or OX40) to T cells. Then, we demonstrate that the combination of PL1-OX40 mRNA and anti-OX40 antibody exhibits significantly improved antitumor activity compared to anti-OX40 antibody alone in multiple tumor models. This treatment regimen results in a 60% complete response rate in the A20 tumor model, with these mice being resistant to rechallenge by A20 tumor cells. Additionally, the combination of PL1-OX40 mRNA and anti-OX40 antibody significantly boosts the antitumor immune response to anti-PD-1 + anti-CTLA-4 antibodies in the B16F10 tumor model. This study supports the concept of delivering mRNA encoding costimulatory receptors in combination with the corresponding agonistic antibody as a strategy to enhance cancer immunotherapy.

EZH2 Inhibition Compromises α4-1BB-Mediated Antitumor Efficacy by Reducing the Survival and Effector Programming of CD8+ T Cells

Enhancer of Zeste Homolog 2 (EZH2) inhibitors (EZH2i) are approved to treat certain cancer types. Previous studies have suggested the potential to combine EZH2i with immune checkpoint blockade targeting coinhibitory receptors like PD-(L)1 and CTLA-4, but whether it can also enhance the activity of agents targeting costimulatory receptors is not known. Here, we explore the combination between EZH2i and an agonist antibody targeting the T cell costimulatory receptor 4-1BB (α4-1BB). Our data show that EZH2i compromise the efficacy of α4-1BB in both CT26 colon carcinoma and in an in vivo protein immunization model. We link this to reduced effector survival and increased BIM expression in CD8+ T cells upon EZH2i treatment. These data support the requirement of EZH2 function in 4-1BB-mediated CD8+ T cell expansion and effector programming and emphasize the consideration that must be given when combining such antitumoral therapies.

566 ATOR-1017, a second generation 4–1BB antibody with potential to enhance efficacy of PD-1 therapies

BackgroundATOR-1017 is a Fcγ-receptor (FcγR) crosslinking dependent agonistic IgG4 antibody targeting the costimulatory receptor 4 1BB, designed for improved tolerability and efficacy. 4-1BB is highly expressed on tumor infiltrating CD8+ T effector cells (T effs) in several cancer indications. By binding to 4-1BB, ATOR-1017 enhances the activity of tumor reactive T effs and NK cells within the tumor and induces a potent anti-tumor response. 4-1BB is a promising candidate for immunotherapy and holds great potential for combination with other immunomodulatory antibodies, targeting e.g. the PD-1 pathway.MethodsHuman 4-1BB knock-in transgenic mice with established murine colon carcinoma MC38 tumors were used to demonstrate anti-tumor efficacy after systemic treatment with ATOR-1017 in combination with anti-PD-1. Further, the effect of combining ATOR-1017 with anti-PD-1 on T cell activation (measured as production of IFNγ) was evaluated in a mixed lymphocyte reaction (MLR) assay with human primary CD4+ T cells and mature monocyte-derived DCs (mDC) expressing endogenous levels of both 4-1BB and PD-1.ResultsATOR-1017 in combination with anti-PD-1 improved survival and reduced tumor growth signifcantly in human 4-1BB knock-in transgenic mice with established tumors compared with each monotherapy alone. The potential for combining ATOR-1017 and PD-1 was further supported by data from a MLR assay demonstrating that the combination of ATOR-1017 with anti-PD-1 induced a more potent CD4+ T cells activation than each monotherapy alone.The functional activation profile of ATOR-1017 is expected to minimize the risk of systemic immune activation and toxicity, by directing a potent immune response to immune cells in tumor tissue and tumor draining lymph nodes. This is supported by early data from the ongoing first-in-human phase I study where ATOR-1017 has been shown to be safe and tolerable.ConclusionsIn summary, these results support further clinical development of ATOR-1017 in combination with PD-1 antibodies. By combining ATOR-1017 with anti-PD-1, tumor infiltrating T cells can be more effectively activated and potentially increase the response rate in multiple indications.Ethics ApprovalAll animal procedures were in accordance to IACUC guidance

Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer

BackgroundSuccessful targeting of solid tumors such as breast cancer (BC) using chimeric antigen receptor (CAR) T cells has proven challenging, largely attributed to the immunosuppressive tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSCs) inhibit CAR T cell function and persistence within the breast TME. To overcome this challenge, we have developed CAR T cells targeting tumor-associated mucin 1 (MUC1) with a novel chimeric costimulatory receptor that targets tumor necrosis factor–related apoptosis-inducing ligand receptor 2 (TR2) expressed on MDSCs.MethodsThe function of the TR2.41BB costimulatory receptor was assessed by exposing non-transduced (NT) and TR2.41BB transduced T cells to recombinant TR2, after which nuclear translocation of NFκB was measured by ELISA and western blot. The cytolytic activity of CAR.MUC1/TR2.41BB T cells was measured in a 5-hour cytotoxicity assay using MUC1+ tumor cells as targets in the presence or absence of MDSCs. In vivo antitumor activity was assessed using MDSC-enriched tumor-bearing mice treated with CAR T cells with or without TR2.41BB.ResultsNuclear translocation of NFκB in response to recombinant TR2 was detected only in TR2.41BB T cells. The presence of MDSCs diminished the cytotoxic potential of CAR.MUC1 T cells against MUC1+ BC cell lines by 25%. However, TR2.41BB expression on CAR.MUC1 T cells induced MDSC apoptosis, thereby restoring the cytotoxic activity of CAR.MUC1 T cells against MUC1+ BC lines. The presence of MDSCs resulted in an approximately twofold increase in tumor growth due to enhanced angiogenesis and fibroblast accumulation compared with mice with tumor alone. Treatment of these MDSC-enriched tumors with CAR.MUC1.TR2.41BB T cells led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared with CAR.MUC1 (469.79±81.46 mm3) or TR2.41BB (434.86±64.25 mm3) T cells alone. CAR.MUC1.TR2.41BB T cells also demonstrated improved T cell proliferation and persistence at the tumor site, thereby preventing metastases. We observed similar results using CAR.HER2.TR2.41BB T cells in a HER2+ BC model.ConclusionsOur findings demonstrate that CAR T cells that coexpress the TR2.4-1BB receptor exhibit superior antitumor potential against breast tumors containing immunosuppressive and tumor promoting MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

33 Development of a clinical ex vivo assay for the assessment of therapeutic CD28 costimulatory pathway engagement

BackgroundPreclinical evidence supports combining checkpoint inhibition (CPI) with T cell costimulatory agonism to improve the breadth and durability of anti-tumor responses relative to CPI alone. Currently, there are a number of therapeutic approaches combining costimulatory receptor agonists (e.g. CD28, 4-1BB, OX40L, etc.) with tumor targeting agents and/or CPI. Identification of a pharmacodynamically-justified therapeutic dose can be challenging because traditional duration of target occupancy does not necessarily correlate with immunological activity in the case of costimulatory molecules, and an ‘always on’ dose risks immune exhaustion. ALPN-202, a variant CD80 vIgD-Fc fusion protein that mediates PD-L1-dependent CD28 costimulation and inhibits the PD-L1 and CTLA-4 checkpoints, is in development for the treatment of multiple advanced malignancies. To assess clinical CD28 agonism in the context of ALPN-202 treatment, we developed a novel, ex vivo whole blood target-dependent costimulation (TDC) assay.MethodsA TDC assay was developed using clinical samples from NEON-1 (NCT04186637), an ongoing dose escalation and expansion clinical trial of ALPN-202 for patients with advanced malignancies. The assay uses patient blood stimulated with paraformaldehyde-fixed, artificial antigen presenting cells (aAPC) expressing both cell-surface anti-CD3 and PD-L1. Pre-dose and end-of-infusion (EOI) blood was drawn from trial participants and co-cultured for 24 hours with the aAPCs in a pre-made assay plate. Plasma was collected and secreted IL-2 was quantified and used as a measure of PD-L1-dependent CD28 costimulation. Nonlinear regression was used to calculate area under the curve (AUC) for each condition, and sample AUC values were compared to a positive control (pre-dose blood stimulated with a fixed concentration of ALPN-202). Serum concentration of ALPN-202 and CD28 target saturation analyses were conducted concurrently to evaluate the exposure-response relationship.ResultsUsing the ex vivo TDC assay, ALPN-202 demonstrated PD-L1-dependent T cell costimulation at all dose levels tested to date in the NEON-1 clinical trial, consistent with preclinical assay development data. Similarly, CD28 target saturation levels on circulating T cells correlated with serum concentration of ALPN-202.ConclusionsWe have developed a novel ex vivo assay to assess induction of PD-L1-dependent CD28 costimulation in a therapeutic setting. This assay has been successfully employed to monitor controlled CD28 costimulation by the CD28 agonist therapeutic candidate ALPN-202, helping to establish a PK/PD relationship that is consistent with preclinical data. More broadly, this type of cell-based, ex vivo TDC assay could be adapted to assess costimulatory receptor engagement, particularly target-dependent costimulation, for other therapeutic agonists in clinical development.Ethics ApprovalThis study was approved by WCG IRB’s Human Subjects Review, approval number: 20211877.

Critical roles of ICOS in controlling Foxp3-expressing T follicular cell subsets during early-stage germinal center reactions

The inducible costimulator (ICOS) is a T cell costimulatory receptor critical for humoral immunity. In mice and humans, ICOS deficiency leads to a lack of T follicular helper (Tfh) cells and protective antibodies. However, the role of ICOS in T follicular regulatory (Tfr) cell generation and function remains poorly understood. Using Foxp3-cre-mediated ICOS knockout (ICOS FC) mice, we show that T regulatory (Treg)-specific ICOS deletion drastically reduces the number of Tfr cells without altering Treg cell numbers. Further, we observed a lowered ratio of antigen-specific germinal center B (GC B) cells and increased anti-nuclear antibodies in ICOS FC mice, suggesting a rise of autoreactive GC B cells. Single-cell transcriptome analysis revealed shifts in transitory Tfr precursor populations in immunized ICOS FC mice. Mechanistically, our data suggest that ICOS promotes the Treg-to-Tfr transition by regulating KLF2 and NFAT2 with downstream impacts on Bcl6 and CXCR5 expression. Importantly, we discovered a Tfr subset originating from Foxp3- precursors by analyzing flow cytometry data of immunized mice in which ICOS expression in Foxp3+ cells was ablated at different stages of GC reactions. Consistently, single-cell transcriptome analysis of global follicular T cells revealed a subset of Tfh-like cells beginning to express Foxp3 at day 6 post-immunization. These Tfh-like Tfr cells appear to promote GC B cell expansion and plasma cell differentiation in an ICOS-dependent manner. Taken together, we propose that Tfr cells derived from natural Tregs suppress the expansion of autoreactive GC B cells whereas Tfh-like Tfr cells promote early-stage GC output. Further, these Tfr subsets depend on ICOS costimulation for their differentiation or function.

Defining the RBPome of primary T helper cells to elucidate higher-order Roquin-mediated mRNA regulation

Post-transcriptional gene regulation in T cells is dynamic and complex as targeted transcripts respond to various factors. This is evident for the Icos mRNA encoding an essential costimulatory receptor that is regulated by several RNA-binding proteins (RBP), including Roquin-1 and Roquin-2. Here, we identify a core RBPome of 798 mouse and 801 human T cell proteins by utilizing global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS). The RBPome includes Stat1, Stat4 and Vav1 proteins suggesting unexpected functions for these transcription factors and signal transducers. Based on proximity to Roquin-1, we select ~50 RBPs for testing coregulation of Roquin-1/2 targets by induced expression in wild-type or Roquin-1/2-deficient T cells. Besides Roquin-independent contributions from Rbms1 and Cpeb4 we also show Roquin-1/2-dependent and target-specific coregulation of Icos by Celf1 and Igf2bp3. Connecting the cellular RBPome in a post-transcriptional context, we find contributions from multiple RBPs to the prototypic regulation of mRNA targets by individual trans-acting factors.