IL-15 Trans-presentation Research Articles

IL-1β Induces Human Endothelial Surface Expression of IL-15 by Relieving let-7c-3p Suppression of Protein Translation.

Expression of IL-15 on the surface of human graft endothelial cells (ECs) bound to the IL-15Rα subunit can increase the activation of CTLs, potentiating allograft rejection. Our previous work showed that surface expression of this protein complex could be induced by alloantibody-mediated complement activation through increased IL-1β synthesis, secretion, and autocrine/paracrine IL-1-mediated activation of NF-κB. In this article, we report that cultured human ECs express eight differently spliced IL-15 transcripts. Remarkably, IL-1β does not alter the expression level of any IL-15 transcript but induces surface expression independently of RNA polymerase II-mediated transcription while requiring new protein translation. Mechanistically, IL-1β causes an NF-κB-mediated reduction in the level of microRNA Let-7c-3p, thereby relieving a block of translation of IL-15 surface protein. Let7c-3p anti-miR can induce EC surface expression of IL-15/IL-15Rα in the absence of complement activation or of IL-1, enabling IL-15 transpresentation to boost CD8 T cell activation. Because of the complexity we have uncovered in IL-15 regulation, we recommend caution in interpreting increased total IL-15 mRNA or protein levels as a surrogate for transpresentation.

Abstract LB072: Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans

Abstract Introduction: We have previously shown the successful engineering of membrane-bound IL15 (mbIL15)-expressing tumor-infiltrating lymphocytes (TIL; cytoTIL15™ cells) from solid tumors such as melanoma, which are CD8+ enriched TIL exhibiting enhanced persistence and anti-tumor activity compared with unengineered TIL (SITC 2022, 2023; clinical candidate OBX-115: NCT05470283). The mbIL15 expressed by cytoTIL15 cells is regulated via a carbonic anhydrase 2 (CA2) drug-responsive domain, which allows expression to be induced upon delivery of the FDA-approved small molecule ligand acetazolamide (ACZ) and was designed to engage the IL2Rβ and IL2Rγ receptors in both cis and in trans. Using natural killer (NK) cells, which are potently responsive to soluble, trans-presented IL15, as an experimental model system, we examined the potential of mbIL15 on cytoTIL15 cells to potentiate activity in both cis and trans. Methods: cytoTIL15 cells and unengineered TIL were generated as described previously (SITC 2022, 2023). Cells were assessed for expression of mbIL15 and phosphorylation of downstream signaling molecules STAT5 and S6 by flow cytometry, and co-cultured with allogeneic healthy donor NK cells for 1-10 days with and without transwell inserts. Functional effects of cytoTIL15 cells were compared to those of soluble recombinant IL15. Supernatant was obtained from co-cultures for assessment of cytokine production. Results: cytoTIL15 cells demonstrated robust ACZ-dependent mbIL15 expression, and ACZ dosing regulated mbIL15 activity in cis by potentiating the phosphorylation of STAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.012) and S6 (geoMFI with 0 vs. 25 µM ACZ, p=0.016). Upon removal of ACZ, mbIL15 expression and pSTAT5 and pS6 returned to basal levels within 24 hours. mbIL15 expressed on the surface of cytoTIL15 cells could also activate cells in trans in an ACZ-dependent titratable manner. Specifically, co-culture of cytoTIL15 cells with NK cells led to an ACZ-dependent increase in pSTAT5 (geoMFI with 0 vs. 25 µM ACZ, p=0.038) in NK cells, which was not seen in co-cultures of unengineered TIL with NK cells. Additionally, NK cells showed a similar cytokine production profile when exposed to mbIL15 by cytoTIL15 cells as with soluble, trans-presented IL15. NK cell transactivation relied on direct cell contact with cytoTIL15 cells, as magnitudes of phosphorylation were decreased in co-cultures with NK and cytoTIL15 cells separated by a transwell membrane (pSTAT5 geoMFI p=0.008). Conclusions: cytoTIL15 cells demonstrate functional engagement of cytokine receptors via mbIL15 in both cis and in trans, including phosphorylation of STAT5 and S6. Taken together, these data highlight the potential for robust activity of cytoTIL15 cells through potentiation of ACZ-dependent mbIL15 activity in both cis and trans. Citation Format: Rachel Burga, Gauri Kulkarni, Zheng Ao, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with membrane-bound IL15 (cytoTIL15 cells) exhibit pharmacologically regulatable signal transduction in cis and trans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB072.

P40 homodimers bridge ischemic tissue inflammation and heterologous alloimmunity in mice via IL-15 transpresentation.

Virus-induced memory T cells often express functional cross-reactivity, or heterologous immunity, to other viruses and to allogeneic MHC molecules that is an important component of pathogenic responses to allogeneic transplants. During immune responses, antigen-reactive naive and central memory T cells proliferate in secondary lymphoid organs to achieve sufficient cell numbers to effectively respond, whereas effector memory T cell proliferation occurs directly within the peripheral inflammatory microenvironment. Mechanisms driving heterologous memory T cell proliferation and effector function expression within peripheral tissues remain poorly understood. Here, we dissected proliferation of heterologous donor-reactive memory CD8+ T cells and their effector functions following infiltration into heart allografts with low or high intensities of ischemic inflammation. Proliferation within both ischemic conditions required p40 homodimer-induced IL-15 transpresentation by graft DCs, but expression of effector functions mediating acute allograft injury occurred only in high-ischemic allografts. Transcriptional responses of heterologous donor-reactive memory CD8+ T cells were distinct from donor antigen-primed memory CD8+ T cells during early activation in allografts and at graft rejection. Overall, the results provide insights into mechanisms driving heterologous effector memory CD8+ T cell proliferation and the separation between proliferation and effector function that is dependent on the intensity of inflammation within the tissue microenvironment.

IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

CD40 is a central costimulatory receptor implicated in productive antitumor immune responses across multiple cancers, including bladder cancer. Despite strong preclinical rationale, systemic administration of therapeutic agonistic antibodies targeting the CD40 pathway has demonstrated dose-limiting toxicities with minimal clinical activity, emphasizing an important need for optimized CD40-targeted approaches, including rational combination therapy strategies. Here, we describe a role for the endogenous IL-15 pathway in contributing to the therapeutic activity of CD40 agonism in orthotopic bladder tumors, with upregulation of transpresented IL-15/IL-15Rα surface complexes, particularly by cross-presenting conventional type 1 DCs (Dendritic Cells), and associated enrichment of activated CD8 T cells. In bladder cancer patient samples, we identify DCs as the primary source of IL-15, although they lack high levels of IL-15Rα at baseline. Using humanized immunocompetent orthotopic bladder tumor models, we demonstrate the ability to therapeutically augment this interaction through combined treatment with anti-CD40 agonist antibodies and exogenous IL-15, including the fully-human Fc-optimized antibody 2141-V11 currently in clinical development for the treatment of bladder cancer. Collectively, these data reveal an important role for IL-15 in mediating antitumor CD40 agonist responses in bladder cancer and provide key proof-of-concept for combined use of Fc-optimized anti-CD40 agonist antibodies and agents targeting the IL-15 pathway. These data support expansion of ongoing clinical studies evaluating anti-CD40 agonist antibodies and IL-15-based approaches to develop combinations of these promising therapeutics for the treatment of patients with bladder cancer.

Inhibition of TCR-MHC-II interactions abrogates Treg-mediated immune suppression and augments anti-tumor immunity.

Abstract CD4 +Foxp3 +Tregs play a non-redundant role in controlling autoimmunity. Continuous TCR signaling and the availability of exogenous IL-2 from CD4 +effectors regulate the size of the Treg pool and their suppressive function in the steady state. Homeostatic proliferation of Tregs and memory phenotype (MP) CD4 +Foxp3 −T cells depends on CD80/CD86-CD28 signaling. Acute blockade of TCR-MHC-II interactions in vivo by anti-MHC-II mAb significantly enhances memory phenotype (MP) Treg, CD4 +, and CD8 +T cell expansion by a CD28-dependent pathway. IL-15 trans presentation by APC also contributes to Treg, MP CD4 +and MP CD8 +proliferation following blockade of TCR-MHC-II interaction by a CD122-dependent pathway. Combined CD28 activation and IL-15 stimulation together with IL-2 deprivation secondary to MHCII-TCR blockade result in decreased expression of CD25, but enhancement of Foxp3 expression by Treg. In addition, Treg exhibit decreased pAKT/NFAT signaling, but enhanced pSTAT5/mTOR signaling. Mice transplanted with several different transplantable tumors manifest enhanced tumor immunity in the presence of MHCII-TCR inhibition. Our results demonstrate that in the absence of TCR-MHC-II interactions, Treg proliferate, but lose their identity and manifest disabled suppressor function resulting in enhanced innate and adaptive immunity against cancer progression. This work was supported by the Intramural Research Program of NIAID, NIH. This work was supported by the Intramural Research Program of NIAID, NIH.

Tissue-resident memory T cell maintenance during antigen persistence requires both cognate antigen and interleukin-15.

Our understanding of tissue-resident memory T (TRM) cell biology has been largely developed from acute infection models in which antigen is cleared and sterilizing immunity is achieved. Less is known about TRM cells in the context of chronic antigen persistence and inflammation. We investigated factors that underlie TRM maintenance in a kidney transplantation model in which TRM cells drive rejection. In contrast to acute infection, we found that TRM cells declined markedly in the absence of cognate antigen, antigen presentation, or antigen sensing by the T cells. Depletion of graft-infiltrating dendritic cells or interruption of antigen presentation after TRM cells were established was sufficient to disrupt TRM maintenance and reduce allograft pathology. Likewise, removal of IL-15 transpresentation or of the IL-15 receptor on T cells during TRM maintenance led to a decline in TRM cells, and IL-15 receptor blockade prevented chronic rejection. Therefore, antigen and IL-15 presented by dendritic cells play nonredundant key roles in CD8 TRM cell maintenance in settings of antigen persistence and inflammation. These findings provide insights that could lead to improved treatment of chronic transplant rejection and autoimmunity.

Correction: IFN-γ Induces IL-15 Trans-Presentation by Epithelial Cells via IRF1.

Correction: IFN-γ Induces IL-15 Trans-Presentation by Epithelial Cells via IRF1.

Abstract CT187: Phase 1 Trial of RTX-240, allogeneic red blood cells engineered to express 4-1BBL and trans-presented IL-15, in patients (Pts) with advanced solid tumors

Abstract Background: RTX-240 is an allogeneic cellular therapy genetically engineered to express high copy numbers of trimeric 4-1BBL and IL-15/IL-15Rα fusion proteins on the RBC surface membrane. RTX-240 is designed to activate and expand CD8+ memory T cells and NK cells and is restricted to the normal biodistribution of red blood cells to mitigate toxicity. Safety, pharmacodynamic (PD) effects, pharmacokinetics (PK) and preliminary efficacy of RTX-240 were assessed in a Phase 1 study of pts with solid tumors. Methods: Pts with relapsed/refractory solid tumors not eligible for standard therapy were treated with monotherapy RTX-240 in 10 cohorts utilizing 6 dose levels (1x108-5x1010 cells per dose) and 4 different schedules until disease progression or unacceptable toxicity. Peripheral blood and tumor tissue were collected at baseline and on-treatment. Results: As of Dec 3, 2021, 30 pts with solid tumors (median age 58 yr, range 23-80, 17 males, 20 ECOG 1), were treated with single agent RTX-240. Pts had previously received a median of 3 therapies (range, 1-9); 21 pts had received prior PD-1/PD-L1 inhibitor (i) therapy. Common tumor types included NSCLC (n=7), melanoma (n=7), colorectal/lower GI (n=4) and RCC (n=3). Per RECIST v1.1, a confirmed partial response (PR) was observed in 1 pt with anal carcinoma and an unconfirmed PR in a uveal melanoma, both of whom had been previously treated with a PD-1. Disease control (PR or stable disease > 12 weeks) occurred in 11/30 pts (37%), of which 7 had received a prior PD-1 for a median of 8 months. Peripheral blood PD studies demonstrate activation, expansion, and cytotoxic potential (GzB) of both NK and memory CD8+ T cells. Every pt with evaluable samples (n=29) had an increase in at least one of the activation or expansion parameters, and the majority had an increase in all. There was also a clear dose response effect in NK cell numbers and trends in other PD markers. Immune activation was observed by increases in plasma IFN-γ in a subset of patients. On-treatment biopsies in a subset (3/5) of evaluable pts show infiltration of NK and/or T cells into the tumor microenvironment (TME) after RTX-240 dosing. No DLTs or related grade (Gr)>3 AEs were seen. The most common treatment-related AEs (Gr 1-2) were fatigue (n = 6); chills, nausea, decrease in appetite, arthralgia (n =3); and fever, myalgia, dysgeusia, and hyperhidrosis (n=2). Additional Gr 2 irAEs include pneumonitis, adrenal insufficiency, hypothyroidism, and worsening transaminitis. Conclusions: RTX-240 is well tolerated at all tested doses, schedules and routes. The drug leads to activation, expansion and trafficking of two target cells (memory CD8+ T cells and NK cells) into the TME, exhibits a clear dose response effect with NK cells and demonstrates preliminary evidence of anti-tumor activity. Dose escalation continues and a PD-1 inhibitor combination arm is also enrolling. Citation Format: Omid Hamid, Melissa L. Johnson, Jason Luke, Richard T. Maziarz, Jamie Merchan, Emerson E. Lim, Sandip P. Patel, Geoffrey M. Kuesters, Iga Sienczylo, Gilad Gordon, Karen Campbell, Kristin Horton, Laurence Turka, Alexander I. Spira. Phase 1 Trial of RTX-240, allogeneic red blood cells engineered to express 4-1BBL and trans-presented IL-15, in patients (Pts) with advanced solid tumors [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 CT187.

Abstract 2864: A CLEC12A immunocytokine with target cell-restricted IL-15 activity shows a favorable toxicity profile and high potency in AML

Abstract IL-15, often referred to as the “anti-cancer cytokine”, potently stimulates proliferation and activation of NK and T cells, but unlike its close relative IL-2, does neither promote activation-induced death of lymphocytes nor activity of immunoinhibitory Tregs. So far, short half-life, poor accumulation at the tumor site and severe toxicity upon systemic application limit IL-15 efficacy in patients. Fusion of IL-15 to antibodies directed to tumor antigens (classical immunocytokines, ICs) improves on accumulation at the tumor site and pharmacokinetics. However, since the activity of the cytokine moiety within classical ICs does not depend on antigen binding, the application of clinically effective doses is still prevented by toxicity due to unspecific immune activation. To overcome this problem, we took advantage of the unique mechanism of action of IL-15 which stimulates IL-15Rβ/γ on NK and T cells as a membrane-bound complex with IL-15Rα on monocytes and DCs (trans-presentation). We used an Fc-optimized antibody directed to CLEC12A, a surface antigen abundantly expressed on AML cells and leukemic stem cells but not on healthy stem cells. This antibody termed 33C2-SDIE was fused to an IL-15E46K mutant with abolished binding to IL-15Rα, allowing to substitute physiological trans-presentation of IL-15 by binding of the construct to its tumor-expressed target. Antigen-specific binding of the resulting modified immunocytokine (MIC12) was confirmed using multiple CLEC12A-expressing cell lines and primary AML samples from patients. Functional analysis of activation, cytokine release, and target cell lysis demonstrated that MIC proteins, in contrast to classical IC, stimulate cytotoxic lymphocytes in a highly target cell-restricted manner, allowing for the desired reduction of unspecific immune activation. At the same time, significantly superior NK cell reactivity against AML cells as compared to even the Fc-optimized antibody was observed. Both, IL-15 signaling and engagement of Fc-receptors by the optimized Fc-domain were found to be essential for optimal activity of our MIC constructs. Importantly, only treatment with MIC12 was capable to induce NK cell proliferation, which is required to overcome unfavorable target to effector ratios that prevail in overt cancer disease. In summary, our novel CLEC12A-targeting immunocytokine allows for tumor-restricted stimulation of cytotoxic lymphocytes and reduced toxicity while displaying superior anti-leukemic activity and constitutes a promising compound for the treatment of AML. Citation Format: Boris Klimovich, Leonard Anton, Yangmi Lim, Jonghwa Won, Anna Chashchina, Martin Pflügler, Latifa Zekri, Gundram Jung, Helmut R. Salih. A CLEC12A immunocytokine with target cell-restricted IL-15 activity shows a favorable toxicity profile and high potency in AML [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 2864.

Regulatory T cell coordination of the mucosal NK cell response during viral infection.

Abstract In many infections the mucosal barrier is the site of pathogen exposure, yet the regulation of mucosal immunity is not well understood. Herpes simplex virus-2 (HSV-2) is one of the most prevalent mucosal infections, making this disease an ideal model to study mucosal anti-viral immune response. My goal is to define how regulatory T cells (Tregs) shape anti-viral immune responses through modulation of natural killer (NK) cells during mucosal virus infection. To accomplish my goal, I utilized a mouse model of intravaginal HSV-2 infection. NK cell phenotypes were examined using high parameter flow cytometry. To define how transient Treg depletion during HSV-2 infection impacts NK cells, I utilized Foxp3DTR mice. Treg depletion led to increased maturation of NK cells in the vaginal tract (VT). KLRG1 is associated with NK cell maturation and is a marker of terminally mature NK cell subsets. I found a significant increase in the frequency and total number of KLRG1+ NK cells in the VT two days after HSV-2 infection in the absence of Tregs. I further analyzed NK cell maturation using the expression of the two surface markers CD11b and CD27 and found a significant increase in CD11b+CD27+ NK cells after Treg depletion. This subset of NK cells is known to be significant producers of pro-inflammatory cytokines. The cytokine IL-15 is necessary for NK cell development and maturation. I found that in the VT, CD11b+Ly6C+ monocyte-derived DCs display increased IL-15 transpresentation after Treg depletion. My data suggests that Tregs have a modulatory effect on NK cell development in the VT as Treg depletion leads to altered NK cell maturation and development. This demonstrates the role of Tregs in mediating an effective immune response while limiting immune pathology. Supported by grants from NIH (R01 AI141435-01, T32 AI 7509-21)

A novel fusion protein scaffold 18/12/TxM activates the IL-12, IL-15, and IL-18 receptors to induce human memory-like natural killer cells

Natural killer (NK) cells are cytotoxic innate lymphoid cells that are emerging as a cellular immunotherapy for various malignancies. NK cells are particularly dependent on interleukin (IL)-15 for their survival, proliferation, and cytotoxic function. NK cells differentiate into memory-like cells with enhanced effector function after a brief activation with IL-12, IL-15, and IL-18. N-803 is an IL-15 superagonist composed of an IL-15 mutant (IL-15N72D) bound to the sushi domain of IL-15Rα fused to the Fc region of IgG1, which results in physiological trans-presentation of IL-15. Here, we describe the creation of a novel triple-cytokine fusion molecule, 18/12/TxM, using the N-803 scaffold fused to IL-18 via the IL-15N72D domain and linked to a heteromeric single-chain IL-12 p70 by the sushi domain of the IL-15Rα. This molecule displays trispecific cytokine activity through its binding and signaling through the individual cytokine receptors. Compared with activation with the individual cytokines, 18/12/TxM induces similar short-term activation and memory-like differentiation of NK cells on both the transcriptional and protein level and identical in vitro and in vivo anti-tumor activity. Thus, N-803 can be modified as a functional scaffold for the creation of cytokine immunotherapies with multiple receptor specificities to activate NK cells for adoptive cellular therapy.

IFN-γ Induces IL-15 Trans-Presentation by Epithelial Cells via IRF1.

IL-15 exhibits pleiotropic effects on NK and CD8+ T cells and contributes to host protection or immunopathology during infection. Although both type I IFNs and IFN-γ upregulate IL-15 expression, their effects on IL-15 upregulation and underlying mechanisms have not been compared comprehensively. In addition, little is known about trans-presentation of IL-15 by epithelial cells to lymphocytes. In this study, we analyzed the expression of IL-15 and IL-15Rα in the human hepatocyte-derived Huh-7 cell line after stimulation with IFN-α, IFN-β, or IFN-γ using RT-PCR, flow cytometry, and confocal microscopy. We also performed knockdown experiments to investigate the signaling pathway involved in IL-15 upregulation. IFN-γ more potently upregulated IL-15 expression in Huh-7 cells than IFN-α and IFN-β. Knockdown experiments revealed that IFN-γ- and IFN-β-induced IL-15 expression relied on IFN regulatory factor 1 (IRF1), which is upregulated by STAT1 and IFN-stimulated gene factor 3, respectively. Inhibitor of κB kinase α/β was also involved in IFN-γ-induced upregulation of IL-15. Furthermore, human NK cells were activated by coculture with IFN-γ-treated Huh-7 cells, which was abrogated by knocking down IL-15Rα in IFN-γ-treated Huh-7 cells, indicating that IFN-γ-induced IL-15 on Huh-7 cells activates NK cells via trans-presentation. In summary, our data demonstrate that IFN-γ potently elicits IL-15 trans-presentation by epithelial cells via IRF1. These data also suggest that the IFN-γ-IRF1-IL-15 axis may be a regulatory target for the treatment of diseases with IL-15 dysregulation.

The implications of IL-15 trans-presentation on the immune response.

Interleukin-15 is a pleiotropic cytokine type I four alpha-helical bundle cytokine that along with IL-2, IL-4, IL-7, IL-9, and IL-21 shares the common cytokine receptor γ chain, γc. IL-15 is vital for the development, survival, and expansion of natural killer cells and for the development of CD8+ memory T cells. Whereas other family γc cytokines signal by directly binding to their target cells, IL-15 is distinctive in that it binds to IL-15Rα, a sushi domain containing binding protein that is expressed on a number of cell types, including monocytes and dendritic cells as well as T cells, and then is trans-presented to responding cells that express IL-2Rβ and γc. This distinctive mechanism for IL-15 relates to its role in signaling in the context of cell-cell interactions and signaling synapses. The actions of IL-15 and ways of manipulating its actions to potential therapeutic benefit are discussed.

IL-15 Trans-Presentation Is an Autonomous, Antigen-Independent Process.

IL-15 plays a pivotal role in the long-term survival of T cells and immunological memory. Its receptor consists of three subunits (IL-15Rα, IL-2/15Rβ, and γc). IL-15 functions mainly via trans-presentation (TP), during which an APC expressing IL-15 bound to IL-15Rα presents the ligand to the βγc receptor-heterodimer on a neighboring T/NK cell. To date, no direct biophysical evidence for the intercellular assembly of the IL-15R heterotrimer exists. Ag presentation (AP), the initial step of T cell activation, is also based on APC-T cell interaction. We were compelled to ask whether AP has any effect on IL-15 TP or whether they are independent processes. In our human Raji B cell-Jurkat T cell model system, we monitored inter-/intracellular protein interactions upon formation of IL-15 TP and AP receptor complexes by Förster resonance energy transfer measurements. We detected enrichment of IL-15Rα and IL-2/15Rβ at the synapse and positive Förster resonance energy transfer efficiency if Raji cells were pretreated with IL-15, giving direct biophysical evidence for IL-15 TP. IL-15Rα and MHC class II interacted and translocated jointly to the immunological synapse when either ligand was present, whereas IL-2/15Rβ and CD3 moved independently of each other. IL-15 TP initiated STAT5 phosphorylation in Jurkat cells, which was not further enhanced by AP. Conversely, IL-15 treatment slightly attenuated Ag-induced phosphorylation of the CD3ζ chain. Our studies prove that in our model system, IL-15 TP and AP can occur independently, and although AP enhances IL-15R assembly, it has no significant effect on IL-15 signaling during TP. Thus, IL-15 TP can be considered an autonomous, Ag-independent process.

IMMU-36. B CELL-VACCINE ELICITS LONG TERM IMMUNITY AGAINST GLIOBLASTOMA VIA ACTIVATION AND DIFFERENTIATION OF TUMOR-SPECIFIC CD8+ MEMORY T CELLS

Abstract While immunotherapy is used clinically to treat many cancers, its translation into brain tumors remains elusive. The importance of B cells in cancer immunity has become increasingly clear, and we previously developed a B cell-based cellular vaccine (BVax) against glioblastoma (GBM) by further activating 4-1BBL+ B cells with CD40 agonism and IFNγ. BVax were characterized as professional antigen-presenting cells (APCs) that promote CD8+ T cell migration and persistence in murine tumor-bearing brains. This study seeks to understand the mechanisms underlying BVax-induced CD8+ T cell fitness in the tumor microenvironment. Initial transcriptomic analysis highlighted that Bvax express high levels of IL15Rα, indicating their potential ability to trans-present IL15. Considering IL15 trans-presentation is fundamental in T-cell memory differentiation, we used BVax to induce T cell activation in the presence of exogenous IL15. BVax were better capable of activating antigen-specific CD8+ T cells and promoting a memory phenotype when compared to other professional APCs such as dendritic cells (DCs). T cell receptor (TCR) CDR3β sequencing showed that BVax expanded a number of TCR clones in-vitro that were found in brains of CT2A tumor-bearing mice in-vivo. These BVax-activated CD8+ T cells displayed a stronger antigen recall response and unique metabolic profile compared to DC-activated CD8+ T cells as shown by metabolomic analysis of tumor-infiltrating CD8+ T cells. When comparing the anti-tumor effects of CD8+ T cells activated by various APCs, BVax with exogenous IL15 promoted CD8+ T cells that displayed the most potent cytotoxicity against GBM cells in-vitro. Collectively, this study suggests that the IL15/IL15Rα axis and interactions with CD8+ T cell are key factors of BVax therapy in promoting a robust survival benefit and long-term immunologic memory against GBM in preclinical models. Additionally, the development of T cell therapies based on B cell licensing can be a promising future approach for glioblastoma therapy.

Abstract CT141: A phase 1 trial of RTX-240, an allogeneic engineered red blood cell with cell -surface expression of 4-1BBL and trans-presented IL-15, in patients with advanced solid tumors

Abstract Short title: RTX-240 phase 1 in solid tumorsA phase 1 trial of RTX-240, an allogeneic engineered red blood cell with cell -surface expression of 4-1BBL and trans-presented IL-15, in patients with advanced solid tumorsBackground: T cell checkpoint inhibition has revolutionized the treatment of cancer, however the key challenge in cancer immunotherapy is the development of resistant disease. Immune agonists and cytokines are promising approaches, but have shown limited success in the clinic. RTX-240 is an allogeneic cellular therapy genetically engineered to express high-copy numbers of trimeric 4-1BBL and IL-15/IL-15R fusion proteins on the cell surface. RTX-240 is designed to activate and expand CD8+ memory T cells and NK cells, and is restricted to the normal biodistribution of red blood cells to mitigate toxicity. Safety, pharmacodynamic (PD) effects, pharmacokinetics and preliminary efficacy of RTX-240 were assessed in a phase 1 study in patients (pts) with solid tumors. Methods: Pts with relapsed/refractory solid tumors not eligible for standard therapy were treated in dose escalating cohorts with RTX-240 Q4 or Q6W until disease progression or unacceptable toxicity. An exploratory cohort of IV and intratumoral (IT) dosing was enrolled (QW IV and IT x3 in cycle 1 and Q4W IV in subsequent cycles). Results: As of 11 Dec 2020, 14 pts (median age 55) were treated across 4 dose levels (1x108 to 1x1010 cells) administered IV or IV/IT. Pts had received a median of 3.5 therapies (range, 1-10); 10 pts had received prior PD-1/PD-L1 inhibitor therapy. Common tumor types include colorectal or other GI cancers (n=5) and melanoma (n=5). No patients experienced DLTs and no related grade (gr) >3 AE were observed. The most common related AE (gr 1-2) were fatigue (4 pts); chills, decreased appetite, arthralgia (3 pts each); and fever, myalgia, dysgeusia, nausea and hyperhidrosis (2 pts each). Additional irAE include gr 2 pneumonitis (n=1) and gr 1 elevated ALT/AST (n=1); the majority of irAE were observed in cycle 2 and beyond. RTX-240 was detected at the end of infusion sample in a dose dependent manner. Five pts (Q4W IV dosing) were evaluable for response by RECIST v1.1. A confirmed partial response (PR) was observed in 1 pt with anal cancer. Disease control, including stable disease or PR, occurred in 4/5 pts. PD studies in peripheral blood from all pts indicated increased numbers of NK cells in 12/14 pts (change from baseline, range 1.1-3 fold) and memory CD8+ T cells in 10/14 pts (change from baseline, range 1.2-3.3 fold). Activation of both NK and memory CD8+ T cells was observed by increased HLA-DR expression in 11/14 and 14/14 pts, respectively. Optional on-treatment biopsies are collected and preliminary data in one pt suggests infiltration of activated NK and T cells into the tumor microenvironment following dosing with RTX-240. Conclusions: RTX-240 is tolerable and leads to activation, expansion and trafficking of memory CD8+ T cells and NK cells, with preliminary evidence of anti-tumor activity. Exploration of the dose and schedule are ongoing in this study (NCT04372706). Citation Format: Omid Hamid, Jason Luke, Alexander Spira, Geoffrey M. Kuesters, Iga Sienczylo, Gilad Gordon, Melissa L. Johnson. A phase 1 trial of RTX-240, an allogeneic engineered red blood cell with cell -surface expression of 4-1BBL and trans-presented IL-15, in patients with advanced solid tumors [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 CT141.

Transcription Factors Associated With IL-15 Cytokine Signaling During NK Cell Development.

Natural killer (NK) cells are lymphocytes primarily involved in innate immunity and possess important functional properties in anti-viral and anti-tumor responses; thus, these cells have broad potential for clinical utilization. NK cells originate from hematopoietic stem cells (HSCs) through the following two independent and continuous processes: early commitment from HSCs to IL-15-responsive NK cell progenitors (NKPs) and subsequent differentiation into mature NK cells in response to IL-15. IL-15 is the most important cytokine for NK cell development, is produced by both hematopoietic and nonhematopoietic cells, and functions through a distinct delivery process termed transpresentation. Upon being transpresented to NK cells, IL-15 contributes to NK cell development via the activation of several downstream signaling pathways, including the Ras–MEK–MAPK, JAK–STAT5, and PI3K–ATK–mTOR pathways. Nonetheless, the exact role of IL-15 in NK cell development has not been discussed in a consecutive and comprehensive manner. Here, we review current knowledge about the indispensable role of IL-15 in NK cell development and address which cells produce IL-15 to support NK cell development and when IL-15 exerts its function during multiple developmental stages. Specifically, we highlight how IL-15 supports NK cell development by elucidating the distinct transpresentation of IL-15 to NK cells and revealing the downstream target of IL-15 signaling during NK cell development.

Abstract IA05: Innate immune defenses against cancer: Potential for mobilizing NK cells for cancer immunotherapy

Abstract Immunotherapies that mobilize CD8 T cells have revolutionized cancer therapy in recent years. A likely limitation of CD8 T cells in immunotherapy is that many tumors display few neoantigens and/or lack MHC I expression. Natural killer cells have emerged as exciting targets for new generations of immunotherapeutics. We investigated innate cues necessary for mobilizing spontaneous anti-tumor responses against tumors in mice and identified the cGAS-STING pathway as a critical pathway that is activated by cancer cells and mobilizes both NK cell and T cell mediated tumor rejection. Cyclic dinucleotides (CDNs) are STING agonists that, when injected into tumors, superactivate cellular immunity. We investigated the efficacy of CDNs in mobilizing anti-tumor activity in 6 independent gene edited models of MHC I-deficient tumors. Due to the absence of MHC I, CD8 T cells played no role in these responses. When transplanted syngeneic tumors of all 6 models were established and then treated intratumorally with CDNs (a single treatment, or in some cases 3 early treatments), tumor rejection rapidly ensued, which was NK-dependent and independent of CD8 T cells. In 5 of 6 MHC I-deficient tumor models tested, CDN therapy resulted in the indefinite survival of between 30% and 100% of the animals, with no evidence of residual tumors, suggesting the animals were cured. In one model (MHC I-deficient B16 melanoma cells) early rejection of the tumors was followed by relapse in all of the animals. Tumor rejection induced by CDNs was IFN-I-dependent, and conditional knockout experiments indicated that IFN-I acted directly on NK cells but also indirectly on DCs. After CDN therapy, DCs in the draining lymph nodes upregulated IL-15/IL-15R complexes on the membrane, and tumor rejection was blocked by neutralizing IL-15/IL15R. These data suggested that CDNs induce trans-presentation of IL15 by DCs to NK cells to support the tumor rejection response. CDN-induced tumor rejection was markedly enhanced by combining CDNs with IL-2 superkine (“super2”, in collaboration with C. Garcia), resulting in long-term tumor free survival in nearly all of the mice in the MHC I-deficient B16 and MC-38 models, indicating striking synergy between CDNs and super2. Our results suggest great potential for combining agonists of innate immune responses with superkines for treating cancers that are invisible to CD8 T cells, including MHC I-deficient cancers and potentially cancers with low neoantigen expression. Citation Format: David H. Raulet. Innate immune defenses against cancer: Potential for mobilizing NK cells for cancer immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr IA05.

Complement-activated human endothelial cells stimulate increased polyfunctionality in alloreactive T cells.

Antibody-mediated deposition of complement membrane attack complexes (MACs) on IFN-γ-primed human endothelial cells (ECs) triggers autocrine/paracrine IL-1β-mediated EC activation and IL-15 transpresentation to alloreactive effector memory T cells (TEM ), changes that enable ECs to increase T cell proliferation and cytokine release. Here, we report the use of single-cell microchip 32-plex proteomics to more deeply assess the functionality of the activated T cells and dependence upon EC-derived signals. Compared to control ECs, MAC-activated human ECs increase both the frequency and degree of polyfunctionality among both CD4+ and CD8+ -proliferated TEM , assessed as secreted proteins. IFN-γ and TNF-α remain the predominant cytokines made by alloreactive TEM , but a few CD4+ TEM also made IL-4 while more CD8+ TEM made perforin and granzyme B. Increased polyfunctionality was attenuated by treatment of the MAC-activated ECs with anti-IL-15 blocking antibody more effectively than IL-1 receptor blockade. The increased polyfunctionality of T cells resulting from interactions with MAC-activated ECs may further link binding of donor-specific antibody to T cell-mediated allograft pathologies.

CD8+CD122+ T cell homeostasis is controlled by different levels of IL-15 trans-presentation.

The homeostasis of CD8+CD122+ T cell requires IL-15 trans-presentation. We use Il15ra mutant mice and bone marrow chimeras to assess the role of IL-15 trans-presentation level in CD8+CD122+ T cells homeostasis. We demonstrate that CD8+CD122+ T cells require different levels of IL-15 trans-presentation to support their homeostasis.