884 Engineered toxin body mediated depletion of TIGIT expressing immune cells for cancer immunotherapy

BackgroundTIGIT (T cell immunoreceptor with Ig and ITIM domains) is an exciting novel target for immuno-oncology which functions as an immune checkpoint on multiple immune cell types including memory CD8+, CD4+ Treg, and memory CD4+ cells. TIGIT upregulation on tumor infiltrating lymphocytes (TILs) has been observed in multiple cancer types and contributes to an immunosuppressive tumor microenvironment (TME). Interestingly, TIGIT is commonly co-expressed with PD-1 on Tregs in the TME, tumor antigen specific CD8+ T cells and CD8+ TILs, leading to weakened anti-tumor immune responses.1–2 To date, TIGIT inhibiting monoclonal antibodies (mAb) have shown little activity as a monotherapy in clinical and preclinical studies. 3–4 Therefore, current clinical trials are now focused on combining TIGIT mAbs with known commercial PD-1 or PD-L1 mAbs. A TIGIT-specific engineered toxin body (ETB) represents a wholly new approach to targeting TIGIT expressing cells including those co-expressing TIGIT and PD-1.MethodsETBs targeting TIGIT were designed to deplete TIGIT-expressing TILs, including Tregs, directly in the TME. ETBs are proteins that consist of an antibody fragment genetically fused to a proprietary de-immunized (DI) form of the Shiga-like toxin A subunit (SLTA). These proteins are specific for a cell surface receptor, and function through triggering rapid internalization upon binding, followed by an enzymatic and irreversible termination of ribosomal protein synthesis resulting in cellular apoptosis. Here we provide proof of concept for ETBs as a novel modality for the depletion of TIGIT-expressing immune cells.ResultsTIGIT-targeting ETBs exhibit potent in vitro cytotoxicity of TIGIT over-expressing cell lines (IC50<1nM). These ETBs also lead to apoptotic depletion of ex vivo TIGIT-expressing regulatory T cells (Tregs) from healthy donors. In mixed culture assays, TIGIT ETBs increase the proliferation of TIGIT negative T cells by depleting TIGIT-expressing T cells.ConclusionsStudies to assess pharmacodynamics and efficacy of TIGIT targeting ETBs using a double knock-in (TIGIT and PD-1) mouse tumor model are ongoing, but these early proof of concept in vitro data support the hypothesis that ETBs can deplete TIGIT positive immune cell populations including those co-expressing PD-1. It is possible that targeted TIGIT inhibition through ETB-induced cell death could tip the balance towards tumor regression by eliminating this novel checkpoint (and TIGIT/PD-1 co-expression) at the level of the TME.ReferencesJinhua X, Ji W, Shouliang C, Liangfeng Z. Expression of immune checkpoints in T cells of esophageal cancer patients. Oncotarget 2016;7(39):1–10.Blessin NC, Simon R, Kluth M, Fischer K, et al. Patterns of TIGIT expression in lymphatic tissue, inflammation and cancer. Dis Markers 2019;2019:1–13.Johnston RJ, Comps-Agrar L, Hackney J, Yu X, et al. The immunoreceptor TIGIT regulates anti-tumor and antiviral CD8(+) T effector function. Cancer Cell 2014;26(6):923–927.Bendell JC, Bedrad P, Bang Y-J, LoRusso P, et al. Phase Ia/Ib dose-escalation study of the anti-TIGIT antibody Tiragolumab as a single agent and in combination with atezolizumab in patients with advanced solid tumors. Proceedings: AACR Annual Meeting 2020; April 27–28, 2020 and June 22–24, 2020; Philadelphia, PA.

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737 Inhibition of P21-activated kinase 4 (PAK4) reverts immune exclusion and restores anti-tumor immunity in the tumor microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.737 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A768-A768

Author(s):

Yu 'Jerry' Zhou ◽

Gina Chu ◽

Eleanore Hendrickson ◽

Johnni Gullo-Brown ◽

Brandy Chavez ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

Tumor Cells ◽

Immune Cell ◽

Mouse Tumor ◽

Type I ◽

List Type ◽

P21 Activated Kinase ◽

Immune Exclusion

BackgroundP21-activated kinase 4 (PAK4) is a serine/threonine protein kinase that is mostly expressed in tumor and stroma cells. PAK4 activates tumor WNT/β-catenin pathway and regulates cellular morphology, motility, EMT, cell proliferation and survival. Recent studies also showed that PAK4 can actively exclude T cells from tumors, suggesting that therapeutic inhibition of PAK4 can increase T cell infiltration in tumor microenvironment and overcome resistance to checkpoint inhibitor immunotherapy.1MethodsWe generated PAK4 knockout (KO) clones in human and mouse tumor cells to validate its biology in vitro and in vivo. We also performed pharmacological evaluation of PAK4 inhibition using Pfizer compounds (referred to as 'PAK4i compounds' below) for their potential tumor-intrinsic and immune-regulatory roles.ResultsNanostring, qPCR and RNASeq analysis showed that PAK4 depletion led to increase of cytokine expression in tumor, including conventional dendritic cell (cDC)- recruiting chemokine CCL4, and type I IFN / ISG pathway genes that are associated with MHC upregulation such as CXCL10. In addition, PAK4 KO sensitizes B16F10 tumors to anti-PD-1 treatment and increases infiltration of cDC and T cells in the tumor microenvironment.We also showed that small molecule PAK4i compounds induced more potent cancer cell growth inhibition over treated normal PBMCs. PAK4i compounds also increased immune-activating and decreased immune exclusion genes in B16F10 cells and tumor explants in vitro. Although PAK4 target engagement is demonstrated by CETSA assay, the compound potency on modulating PAK4 downstream Wnt/ β-catenin pathway is low, suggesting that the aforementioned phenotypic changes induced by PAK4i compounds may be partially attributed to other off-target effects.ConclusionsCollectively, our data suggests that genetic depletion or pharmacological inhibition of PAK4 may induce immune-activating cytokine production in tumor cells, revert immune cell exclusion in tumor microenvironment, and synergize with checkpoint blockade therapies. However, further optimization on these PAK4i compounds is needed to improve its specificity on modulating PAK4 enzyme activities.ReferenceAbril-Rodriguez G, Torrejon DY, Liu W, Zaretsky JM, Nowicki TS, Tsoi J, Puig-Saus C, Baselga-Carretero I, Medina E, Quist MJ, Garcia AJ, Senapedis W, Baloglu E, Kalbasi A, Cheung-Lau G, Berent-Maoz B, Comin-Anduix B, Hu-Lieskovan S, CWang CY, Grasso CS & Ribas A. PAK4 inhibition improves PD-1 blockade immunotherapy. Nat Cancer 2020;1:46–58.Ethics ApprovalAll animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Pfizer. Approved protocol # LAJ-2019-01347

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843 Development and engineering of human sialidase for degradation of immunosuppressive sialoglycans to treat cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.843 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A884-A884

Author(s):

Li Peng ◽

Lizhi Cao ◽

Sujata Nerle ◽

Robert LeBlanc ◽

Abhishek Das ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Cells ◽

First Generation ◽

Single Agent ◽

Mouse Tumor ◽

Cytokine Release ◽

Tumor Models ◽

Cell Immunity ◽

Mouse Tumor Models

BackgroundSialoglycans, a type of glycans with a terminal sialic acid, have emerged as a critical glyco-immune checkpoint that impairs antitumor response by inhibiting innate and adaptive immunity. Upregulation of sialoglycans on tumors has been observed for decades and correlates with poor clinical outcomes across many tumor types. We previously showed that targeted desialylation of tumors using a bifunctional sialidase x antibody molecule, consisting of sialidase and a tumor-associated antigen (TAA)-targeting antibody, has led to robust single-agent efficacy in mouse tumor models. In addition to tumor cells, most immune cells present substantially more abundant sialoglycans than non-hematological healthy cells, which may also contribute to immunosuppression. Therefore, we studied the impact of immune cell desialylation and evaluated the therapeutic potential of a newly developed sialidase-Fc fusion (Bi-Sialidase), which lacks a TAA-targeting moiety and consists of engineered human neuraminidase 2 (Neu2) and human IgG1 Fc region, in preclinical mouse tumor models.MethodsThe first generation Neu2 variant was further optimized to improve titers and stability to constructed Bi-Sialidase. Bi-Sialidase’s desialylation potency and impact on immune responses were studied in vitro using various human immune functional assays, including T-cell activation, allogeneic mixed lymphocyte reaction, antibody-dependent cellular cytotoxicity, macrophages polarization/activation, neutrophil activation, and peripheral blood mononuclear cell (PBMC) cytokine release assays. We evaluated its antitumor efficacy in mouse tumor models. Bi-Sialidase’s safety profile was characterized by conducting rat and non-human primate (NHP) toxicology studies.ResultsThe optimized Bi-Sialidase achieved a titer of 2.5 g/L from a 15-day fed-batch Chinese hamster ovary cell culture; in contrast, the wild-type and first-generation Neu2 had no production or a low titer (<0.1 g/L) under similar conditions, respectively. We demonstrated that Bi-Sialidase led to dose-dependent desialylation of immune cells and potentiated T-cell immunity, without impacting NK, macrophage, or neutrophil activation by desialylating immune cells. Activated and exhausted T cells upregulated surface sialoglycans and Bi-Sialidase-mediated desialylation reinvigorated exhausted-like T cells as measured by IFNg production. Bi-Sialidase treatment also enhanced DC priming and activation of naïve T cells by desialylating both T cells and DCs. Furthermore, Bi-Sialidase showed single-agent antitumor activity in multiple mouse tumor models, including MC38, CT26, A20, and B16F10. Importantly, Bi-Sialidase did not cause cytokine release in human PBMC assays and was tolerated to up to 100 mg/kg in rats and NHPs, demonstrating a wide safety margin.ConclusionsBi-Sialidase with an optimized Neu2 offers a novel immunomodulatory approach to enhancing T-cell immunity by desialylating immunosuppressive sialoglycans for cancer treatment.

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Effect of MMP9 inhibition on effector T-cell responses in a PD1-axis refractory model.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.104 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 104-104

Author(s):

Victoria Smith ◽

Vladi Juric ◽

Amanda Mikels-Vigdal ◽

Chris O'Sullivan ◽

Maria Kovalenko ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Tumor Growth ◽

Cell Activation ◽

Immune Cell ◽

Single Agent ◽

Fold Increase ◽

Cytotoxic T Cell

104 Background: Matrix metalloproteinase 9 (MMP9) acts via diverse mechanisms to promote tumor growth and metastasis, and is a key component of the immune-suppressive myeloid inflammatory milieu. We developed a monoclonal antibody (AB0046) that inhibits murine MMP9 and assessed its mechanism of action in immunocompetent mice as a single agent, or in combination with a murine anti-PDL1 antibody. Methods: An orthotopic, syngeneic tumor model (NeuT), which models MMP9-positive myeloid infiltrate, was utilized for efficacy and pharmacodynamic studies involving RNA and T cell receptor (TCR) sequencing, and flow cytometry. Enzymatic analyses were performed on T cell chemoattractant CXCR3 ligands (CXCL9, CXCL10, and CXCL11) which were subsequently evaluated in chemotaxis assays. Results: Anti-MMP9 treatment alone or in combination with an anti-PDL1 antibody decreased primary tumor growth as compared to IgG control-treated animals (56% vs 335% tumor growth increase, p = 0.0005) or anti-PDL1 alone. Profiling of tumors by RNA sequencing revealed that inhibition of MMP9 resulted in elevated expression of genes associated with immune cell activation pathways (Hallmark Interferon Gamma Response, FDR p < 0.001). Treatment with anti-MMP9 and anti-PDL1 antibodies decreased TCR clonality, with evidence of a more diverse TCR repertoire (p = 0.005). Immunophenotyping of tumor-associated T cells by flow cytometry showed that anti-MMP9 and anti-PDL1 co-treatment promoted a 2.8-fold increase in CD3+ cells in tumors (p = 0.01), which was associated with an increase in CD4+ T cells (3.2-fold increase; p = 0.006) and CD8+ T cells (2.8-fold increase; p = 0.013). In contrast, anti-MMP9 and combination treatment resulted in a decrease in tumor-associated regulatory T cells (CD25+ FoxP3+ cells, p = 0.04). MMP9 cleavage of T cell chemoattractant ligands in vitro rendered them functionally inactive for recruitment of activated primary human effector T cells. Conclusions: Inhibition of MMP9 reduces tumor burden and promotes cytotoxic T cell infiltration in a PD1-axis refractory mouse model. The combination of nivolumab and GS-5745, a humanized anti-MMP9 inhibitory antibody, is currently being evaluated in gastric cancer (NCT02864381).

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Key Immune Cell Subsets Are Dysregulated in Patients with Myelofibrosis

Blood ◽

10.1182/blood.v124.21.5580.5580 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5580-5580

Author(s):

Marco Romano ◽

Lucia Catani ◽

Daria Sollazzo ◽

Martina Barone ◽

Margherita Perricone ◽

...

Keyword(s):

T Cells ◽

Immune Cells ◽

Th17 Cells ◽

Immune Cell ◽

Opportunistic Infections ◽

Fungal Infections ◽

High Rate ◽

Healthy Controls ◽

Immune Cell Subsets

Abstract Introduction: Myelofibrosis (MF) is a clonal disorder associated mainly with JAK2V617F and MPL mutations. Recently, a new mutation in the gene encoding calreticulin (CALR) was discovered in the majority of JAK2/MPL negative patients. MF is burdened by a high rate of potentially life-threatening infections. The issue of recurrent and opportunistic infections is increased after the introduction in clinical practice of JAK inhibitors with immunosuppressive activity. However, the role of crucial immune cell subsets is still poorly characterized. Here, we investigated the phenotype/function of selected immune cells in MF. Specifically, we focused on circulating regulatory (Tregs) and IL-17-producing T cells (Th17 cells), monocytes and dendritic cells (DCs). Monocyte-derived DCs were also characterized. Methods: We characterized circulating Th17 cells, Tregs, monocytes and DCs of 17 untreated MF patients and 8 healthy controls (HC) by flow cytometry. Th17 cells were identified as CD4+ CD161+ CD196+ cells while Tregs were enumerated as CD4+ CD25high CD127low T cells. We also tested the in vitro suppressive activity of circulating CD4+ CD25+ Tregs with a mixed leukocyte reaction assay. Two subpopulations of circulating DCs, myeloid CD11c+ and plasmacytoid CD123+cells, were enumerated as well. In addition, after immunomagnetic selection, we tested both phenotype of circulating monocytes and their capacity to differentiate into CD14-derived immature and mature DCs, using a specific cytokines cocktail. JAK2V617F and MPL mutations were detected with RT-PCR while the presence of CALR mutations were tested with Exon 9 Next Generation Sequencing assay. Results: JAK2V617F (11 cases), MPL (3 cases), and CARL (3 cases) mutations were detected. We found that circulating CD4+CD25highCD127low Tregs were reduced in MF patients as compared with healthy controls (p=0.043), although their suppressive ability was maintained. We also found a lower number of circulating Th17 cells (p=0.0026) in MF patients. This finding was particularly evident in JAK2V617F+(p=0.008) and CARL+(p=0.03) patients. Despite their number was in the normal range, circulating monocytes from MF patients showed reduced expression of the CD86 co-stimulatory molecule. Moreover, as compared with the normal counterparts, immature monocytes-derived DCs from patients maintained low CD14 expression without upregulating the CD80 co-stimulatory molecule expression (p=0.0063). Interestingly, at variance with plasmacytoid DCs, a reduced number of circulating myeloid DCs was observed in MF patients as compared with that of HC (p=0.01). Conclusions: Here we demonstrated that specific crucial subsets of immune cells show quantitative and/or qualitative abnormalities in MF patients. These findings may be useful to better understand the increased susceptibility of these patients to infections, since Th17 cells play a role in bacterial and fungal infections while myeloid DCs regulate Th1 activity. Of note, DCs inhibition might result in increased propensity to infections and compromised immune response to cancer.In addition, since monocytes are DC precursors, alterations in their differentiation pathway may contribute to develop defective immune responses. Disclosures Martinelli: NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

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Authentic Patient-Derived Hepatitis C Virus Infects and Productively Replicates in Primary CD4+and CD8+T LymphocytesIn Vitro

Journal of Virology ◽

10.1128/jvi.01790-17 ◽

2017 ◽

Vol 92 (3) ◽

Cited By ~ 4

Author(s):

Georgia Skardasi ◽

Annie Y. Chen ◽

Tomasz I. Michalak

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Lymphocytes ◽

Immune Cells ◽

Immune Cell ◽

Cell Types ◽

Hcv Rna ◽

Biophysical Properties

ABSTRACTAccumulated evidence indicates that immune cells can support the replication of hepatitis C virus (HCV) in infected patients and in culture. However, there is a scarcity of data on the degree to which individual immune cell types support HCV propagation and on characteristics of virus assembly. We investigated the ability of authentic, patient-derived HCV to infectin vitrotwo closely related but functionally distinct immune cell types, CD4+and CD8+T lymphocytes, and assessed the properties of the virus produced by these cells. The HCV replication system in intermittently mitogen-stimulated T cells was adapted to infect primary human CD4+or CD8+T lymphocytes. HCV replicated in both cell types although at significantly higher levels in CD4+than in CD8+T cells. Thus, the HCV RNA replicative (negative) strand was detected in CD4+and CD8+cells at estimated mean levels ± standard errors of the means of 6.7 × 102± 3.8 × 102and 1.2 × 102± 0.8 × 102copies/μg RNA, respectively (P< 0.0001). Intracellular HCV NS5a and/or core proteins were identified in 0.9% of CD4+and in 1.2% of CD8+T cells. Double staining for NS5a and T cell type-specific markers confirmed that transcriptionally competent virus replicated in both cell types. Furthermore, an HCV-specific protease inhibitor, telaprevir, inhibited infection in both CD4+and CD8+cells. The emergence of unique HCV variants and the release of HCV RNA-reactive particles with biophysical properties different from those of virions in plasma inocula suggested that distinct viral particles were assembled, and therefore, they may contribute to the pool of circulating virus in infected patients.IMPORTANCEAlthough the liver is the main site of HCV replication, infection of the immune system is an intrinsic characteristic of this virus independent of whether infection is symptomatic or clinically silent. Many fundamental aspects of HCV lymphotropism remain uncertain, including the degree to which different immune cells support infection and contribute to virus diversity. We show that authentic, patient-derived HCV productively replicatesin vitroin two closely related but functionally distinct types of T lymphocytes, CD4+and CD8+cells. The display of viral proteins and unique variants, the production of virions with biophysical properties distinct from those in plasma serving as inocula, and inhibition of replication by an antiviral agent led us to ascertain that both T cell subtypes supported virus propagation. Infection of CD4+and CD8+T cells, which are central to adaptive antiviral immune responses, can directly affect HCV clearance, favor virus persistence, and decisively influence the development and progression of hepatitis C.

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500 P2RX7 agonist treatment boosts the ability of IL-12-activated CD8+ T cells to infiltrate and control murine melanoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0500 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A535-A535

Author(s):

Kelsey Wanhainen ◽

Stephen Jameson ◽

Henrique Borges Da Silva

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Cd8 T Cells ◽

Mitochondrial Respiration ◽

Immune Cell ◽

List Type ◽

Memory Cd8 T Cells ◽

And Control

BackgroundExtracellular adenosine triphosphate (eATP) is a ‘danger signal’ used to sense cellular damage, and recognized by purinergic receptors in mammals. Among those receptors, P2RX7 is preferentially expressed in immune cells. Notably, we recently discovered that P2RX7 is crucial for the generation and maintenance of long-lived tissue-resident and circulating memory CD8+ T cells.1 2 CD8+ T cell function is fundamental for tumor control, and therapies to harness protective CD8+ T cells that overcome exhaustion are currently in the limelight of anticancer strategies. Given our previous data, and the fact that eATP is abundantly present inside the melanoma microenvironment, we tested whether (a) P2RX7 is required for activated CD8+ T cells to infiltrate and control melanoma upon adoptive cell therapy, and (b) P2RX7 agonism can boost the anticancer capacity of CD8+ T cells.Methods(a) We in vitro-activated WT or P2rx7-/- CD8+ T cells (transgenic for the LCMV epitope gp33-P14 or for the ovalbumin SIINFEKL peptide-OTI) with anti-CD3/CD28/IL-2, ± IL-12, for 72h. Cells were adoptively transferred (single transfer of WT or P2rx7-/- cells) into mice with 7 days after subcutaneous transfer of B16 melanoma encoding gp33 or SIINFEKL. We tracked tumor growth until 60 days or at the appropriate endpoint. In some experiments, we sacrificed recipient mice 7 days after adoptive T cell transfer for immune cell phenotyping. Some parameters (cytokine production, mitochondrial respiration via Seahorse) were measured in in vitro-activated cells. (b) WT and P2rx7-/- cells were activated with anti-CD3/anti-CD28/IL-2, ± Bz-ATP, a P2RX7 agonist. Tumor growth was tracked over time until 60 days or at the appropriate endpoint.ResultsWT and P2RX7-deficient (P2rx7-/-) CD8+ T cells in the absence of IL-12 do not differ in tumor infiltration and/or control. However, P2rx7-/- CD8+ T cells activated in response to IL-12 tertiary stimulus do not control B16 melanomas as well as their WT counterparts. Phenotypically, IL-12-P2rx7-/- CD8+ T cells do not profoundly differ from IL-12-WT CD8+ T cells, except for diminished mitochondrial respiration levels in vitro, and diminished mitochondrial membrane potential (e.g. mitochondrial health) among tumor-infiltrating cells. Strikingly, Bz-ATP treatment increased the mitochondrial activity of WT CD8+ T cells in vitro and in vivo and led to increased B16 infiltration and control, in a P2RX7-dependent manner.ConclusionsWe are currently studying the mechanisms behind the ability of P2RX7 agonists to increase the antitumor function of CD8+ T cells; these are promising results that can lead to a new alternative in immune cell therapies against melanoma.AcknowledgementsWe would like to thank Jane Ding and Lily Qian for technical assistance, and Kristin Hogquist for scientific input.Ethics ApprovalThis study was approved by the IACUC board at the University of Minnesota (IACUC number A3456-01)ReferencesBorges da Silva H, Beura LK, Wang H, Hanse EA, Gore R, Scott MC, Walsh DA, Block KE, Fonseca R, Yan Y, Hippen KL, Blazar BR, Masopust D, Kelekar A, Vulchanova L, Hogquist KA, Jameson SC. The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells. Nature. 2018; 559(7713):264–268.Borges da Silva H, Peng C, Wang H, Wanhainen KM, Ma C, Lopez S, Khoruts A, Zhang N, Jameson SC. Extracellular ATP sensing via P2RX7 promotes CD8+ tissue-resident memory T cells by enhancing TGF-β sensitivity. Immunity 2020;53(1):158–171.

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Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes

Neuro-Oncology Advances ◽

10.1093/noajnl/vdaa056 ◽

2020 ◽

Vol 2 (1) ◽

Cited By ~ 3

Author(s):

Juliana Hofstatter Azambuja ◽

Nils Ludwig ◽

Saigopalakrishna Yerneni ◽

Aparna Rao ◽

Elizandra Braganhol ◽

...

Keyword(s):

T Cells ◽

T Cell Activation ◽

Cd8 T Cells ◽

Immune Cells ◽

Cell Activation ◽

Immune Cell ◽

Size Exclusion ◽

Cell Functions

Abstract Background Glioblastoma is one of the most immunosuppressive human tumors. Emerging data suggest that glioblastoma-derived exosomes (GBex) reprogram the tumor microenvironment into a tumor-promoting milieu by mechanisms that not yet understood. Methods Exosomes were isolated from supernatants of glioblastoma cell lines by size exclusion chromatography. The GBex endosomal origin, size, protein cargos, and ex vivo effects on immune cell functions were determined. GBex were injected intravenously into mice to evaluate their ability to in vivo modulate normal immune cell subsets. Results GBex carried immunosuppressive proteins, including FasL, TRAIL, CTLA-4, CD39, and CD73, but contained few immunostimulatory proteins. GBex co-incubated with primary human immune cells induced simultaneous activation of multiple molecular pathways. In CD8+ T cells, GBex suppressed TNF-α and INF-γ release and mediated apoptosis. GBex suppressed natural killer (NK) and CD4+ T-cell activation. GBex activated the NF-κB pathway in macrophages and promoted their differentiation into M2 cells. Inhibition of the NF-κB pathway in macrophages reversed the GBex-mediated effects. GBex-driven reprogramming of macrophages involved the release of soluble factors that promoted tumor proliferation in vitro. In mice injected with GBex, the frequency of splenic CD8+ T cells, NK cells, and M1-like macrophages was reduced, while that of naïve and M2-like macrophages increased (P < .05). Conclusions GBex reprogrammed functions of all types of immune cells in vitro and altered their frequency in vivo. By creating and sustaining a highly immunosuppressive environment, GBex play a key role in promoting tumor progression.

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Therapy-Inducible Senescence Activates Both Innate and Adaptive Immune Mechanisms That Control Lymphoma Growth In Vivo

Blood ◽

10.1182/blood.v118.21.107.107 ◽

2011 ◽

Vol 118 (21) ◽

pp. 107-107

Author(s):

Jan Dörr ◽

Selina Keppler ◽

Maja Milanovic ◽

Simone Spieckermann ◽

Peter Aichele ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Immune Cells ◽

Immune Cell ◽

Tumor Site ◽

Lymphoma Cells ◽

Host Interactions

Abstract Abstract 107 Introduction: Premature senescence is a cellular failsafe mechanism which is induced upon various cellular insults, such as oncogene activation or exposure to DNA damaging chemotherapy. It suppresses tumor formation and acts as a barrier to tumor progression in vivo. In contrast to apoptotic cells, senescent cells are viably arrested in the G1 phase of the cell cycle. They continue to take up nutrients and interact with tumor and host cells. To what extent senescent cells alter the tumor environment and tumor-host interactions remains largely unsolved. Here, we analyze lymphoma cells with defined genetic lesions, e.g. deletion of the histone H3 lysine 9 methyltransferase Suv39h1 (controlling senescence) and p53 (mediating both apoptosis and senescence), for their influence on immunological tumor-host interactions as a consequence of therapy-induced senescence (TIS) in the Eμ-myc mouse lymphoma model. Our data demonstrate for the first time a senescence-primed T-cell response against lymphoma cells in vitro and in vivo. Methods: Lymphoma cells (LCs) from different genetic were retrovirally transduced with the bcl2 gene to block apoptosis. Subsequently, they were treated with the DNA damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in normal immunocompetent mice in vivo. Therapy-inducible senescence (TIS) was detected based on senescence-associated b-galactosidase activity (SA-b-gal), Ki67 staining and BrdU incorporation. The cytokine profile of senescent LCs was analysed by gene expression and protein arrays. Infiltration and activation of immune cells in TIS lymphomas was analysed by immunohistochemistry and flow cytometry with leukocyte-specific antibodies. Immune responses elicited upon TIS induction in vivo were further analysed in gld (generalized lymphoproliferative disease) mice, which lack functional FasL and by systemic depletion of macrophages after clodronate administration. Pharmaceutical inhibitors of FasL and perforin and IFNg knockout mice were used to analyze T-cell mediated cytotoxity in vitro. Results: TIS lymphoma cells, but not Suv39h1- or p53-deficient LCs, upregulate the secretion of pro-inflammatory cytokines, such as IL6 and IL12, with pro-inflammatory on tumor and bystander cells. In vivo, TIS correlates with the attraction of immune cells, particularly macrophages and T cells, to the tumor site. Senescent LCs became sensitive to both macrophage engulfment and death receptor (Fas)-mediated apoptosis. Activation of both CD4 and CD8 T cells leads to production of IFNg and clearing of senescent cells. Clearance can be attenuated by systemic depletion of macrophages and interference with T cell-mediated programmed cell death. T-cells specifically primed by TIS cells in vivo potently killed both senescent and proliferating LCs after isolation and co-incubation in vitro. In vivo clearance of TIS LCs was attenuated by systemic depletion of macrophages or by interference with T-cell-mediated programmed cell death. Lymphoma-bearing gld mice presented with a reduced overall survival when compared to wild-type host mice. Discussion: This study demonstrates that therapy-induced senescence drives a profound remodeling of the tumor site after therapy and unveils functional interactions of senescent LCs with different immune cell subsets in vitro and in vivo. Senescent cells secrete a cytokine program, which stimulates immune cell attraction and an adaptive and presumably lastingly protective immune response. Thus, TIS is a highly dynamic and interdependent process whose paracrine effects and immune cell interactions account for regression of the senescent mass and present an attractive target network for novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

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MDNA11 is a long-acting IL-2 superkine that potentiates durable immune response in mouse tumor models and non-human primates.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e14546 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e14546-e14546

Author(s):

Minh D. To ◽

Fahar Merchant ◽

Nina Merchant

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cell Activation ◽

Single Agent ◽

Mouse Tumor ◽

Tumor Models ◽

Toxicology Study ◽

Long Acting ◽

Mouse Tumor Models

e14546 Background: The efficacy and safety of recombinant human IL-2 (rhIL-2; Proleukin) to treat certain cancers is limited by a short half-life, marked toxicity and selective high affinity binding to IL2Ra over IL2Rb, resulting in preferential activation of suppressive Tregs. In contrast, MDNA11 has been engineered as a long-acting IL-2 superkine with high affinity IL2Rb receptor selectivity, resulting in preferential anti-cancer effector immune cell activation. Methods: MDNA11 was characterized in both in vitro and in vivo studies including assessment of receptor binding kinetics using BLI/Octet, receptor-mediated signaling in human PMBCs, efficacy in syngeneic mouse tumor models including memory response, as well as safety and PK/PD assessments in non-human primates (NHP). Results: Unlike rhIL-2, MDNA11 does not bind to human IL2Ra but demonstrates a 30-fold higher affinity binding to human IL2Rb. This selectivity resulted in enhanced in vitro STAT5 signaling in human NK and resting CD8 T cells with diminished signaling in Tregs; validation studies in humanized mice are ongoing. In CT26 and MC38 syngeneic tumor models, MDNA11 demonstrates potent and durable efficacy as monotherapy following a Q1W dose schedule for 2 weeks. Synergy with anti-PD1 and anti-CTLA4 immune checkpoint inhibitors (ICIs) was observed and a robust immune memory response developed in all mice with complete tumor clearance. These mice were protected against relapse and tumor re-challenges for up to 8 months without any further treatment, and showed the presence of antigen-specific CD8 T cells. In binding studies with IL-2 receptors of different species, MDNA11 showed highly similar affinity towards human and cynomolgus IL2Rb, confirming the latter as a highly relevant model for toxicology study. MDNA11 was well tolerated in cynomolgus monkeys up to 0.6 mg/kg, while inducing durable (≥10 days) proliferation and expansion of NK and CD8 T cells. Effects on Tregs were minimal and there was no eosinophilia and hypotension (associated with vascular leak syndrome). At high doses of MDNA11, the most common clinical observations were transient loss of appetite and diarrhea. There was modest increase in levels of IFNg and TNFa, but no sign of cytokine release syndrome. Dosing did not trigger development of anti-drug antibodies or histopathologic evidence of pulmonary edema (a major IL-2 induced toxicity). Conclusions: MDNA11 is a long-acting IL-2 superkine that exhibits robust efficacy in mouse tumor models as a single agent and was synergistic in combination with ICIs (anti-CTLA4 and anti-PD1). In NHP, MDNA11 demonstrates selective immune effector cell activation and a favorable safety profile. These data constitute a strong framework for the design of a pivotal GLP toxicology study to further support the planned clinical study of MDNA11 either as a single agent or in combination with ICIs.

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304 Antagonistic antibodies targeting LAIR1 enhance T lymphocyte activation and promote inflammatory phenotypes in myeloid cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.304 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A327-A327

Author(s):

Tao Huang ◽

Caroline Bonnans ◽

Maria Jose Costa ◽

Azita Tabrizi ◽

Jing-Tyan Ma ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Protein Expression ◽

T Cell Activation ◽

Immune Cells ◽

Myeloid Cells ◽

Lymphocyte Activation ◽

List Type

BackgroundThe Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR1) is an immune inhibitory transmembrane glycoprotein expressed on lymphocytes and myeloid cells. The known ligands for LAIR1 are proteins containing collagen-like domains including collagen, complement component 1q (C1q), and stromal protein Colec12.1 2 3 Myeloid-derived suppressor cells (MDSC), tumor associated macrophages (TAMs), as well as collagens, are important contributors of the immune-suppressive tumor microenvironment, and LAIR1 expression is negatively correlated with patient survival in many solid tumors.4 These findings prompt us to investigate LAIR1 as a novel immuno-oncology target in collagen-rich tumors. Utilizing LAIR1 antagonist antibodies, we aim to mobilize anti-tumor immunity by changing the collagen-induced tolerogenic state of the immune cells into proinflammatory.MethodsThe mRNA expression levels of LAIR1, collagen, and C1q in diverse human cancers were analyzed using the TCGA database. LAIR1 protein expression on tumor infiltrated immune cells were measured by flow cytometry. Human tumor samples were obtained from Cooperative Human Tissue Network (CHTN) by the National Cancer Institute (NCI). Purified human T cells from healthy donors were stimulated with immobilized anti-CD3 in the presence of plate-coated human collagen I. Human monocyte-derived macrophages and dendritic cells (DC) were differentiated with M-CSF+IL-4 or GM-CSF+IL-4, respectively. Immune cell phenotypes were assessed by flow cytometry and cytokine secretion by Luminex.ResultsAnalysis of the TCGA database using signature genes specific to macrophages, T cells, DCs, and natural killer (NK) cells demonstrate that LAIR1 is highly expressed in most macrophage-infiltrated tumors and certain T cell-enriched tumors. LAIR1 and collagen are co-expressed at high levels in multiple macrophage-enriched tumors. Flow cytometry analysis of infiltrated immune cells from fresh tumor tissues showed that the highest level of LAIR1 protein expression was detected on TAMs, followed by monocytes, monocytic MDSCs, DCs, and lymphocytes. In vitro, LAIR1 antagonizing antibodies enhanced T-cell activation, proliferation, and IFNγ and TNFα production in comparison to isotype controls in the presence of collagen. Blocking LAIR1 interaction with collagen also decreased the expression of M2 markers such as PD-L1 and CD209 on monocyte-derived M2 macrophages. Additionally, treatment of monocyte-derived DCs by these antibodies increased the expression of the co-stimulatory protein CD86 and promoted the release of IL-12, a crucial cytokine for lymphocyte activation.ConclusionsThese in vitro data suggest that LAIR1 blockade could potentially reverse T-cell and myeloid immunosuppression mediated by collagen, demonstrating the therapeutic potential of anti-LAIR1 antagonistic antibodies.ReferencesMeyaard L. The inhibitory collagen receptor LAIR-1 (CD305). J Leukoc Biol 2008;83:799–803.Son M, et al. C1q limits dendritic cell differentiation and activation by engaging LAIR-1. PNAS 2012;109:3160–3167.Keerthivasan S, et al. Homeostatic functions of monocytes and interstitial lung macrophages are regulated via collagen domain-binding receptor LAIR1. Immunity 2021;54:1511–1526.Xu L, et al. Cancer immunotherapy based on blocking immune suppression mediated by an immune modulator LAIR-1. OncoImmunology 2020;9:e17404771–e17404779.

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