Anlotinib induces a T cell-inflamed tumor microenvironment by facilitating vessel normalization and enhances the efficacy of PD1 checkpoint blockade in neuroblastoma

Abstract BACKGROUND Immunotherapy has demonstrated efficacy in several cancers but has shown only modest effects in Glioblastoma (GBM). This is linked to the anti-inflammatory nature of the tumor microenvironment (TME) and the pro-tumor functions of brain native cells. Targeting stromal cells, such as tumor associated macrophages (TAMs) and neurons, is a promising approach. Re-analysis of human and murine brain single cell-RNAseq (scRNAseq) datasets shows Calmodulin Dependent Kinase Kinase 2 (CaMKK2) is highly expressed in both neurons and TAMs. Loss of CaMKK2 polarizes TAMs to an immunostimulatory phenotype and reduces pro-tumor neuronal functions. Thus, we hypothesize that CaMKK2 promotes the pro-tumor nature of the GBM TME and immunotherapy resistance. RESULTS Murine GBM was orthotopically implanted into wild-type and CaMKK2-/- mice. CaMKK2-/- mice exhibited significantly prolonged survival. To determine if anti-tumor immune function was enhanced, we probed the TME using flow cytometry and scRNAseq. CaMKK2-/- mice showed increased abundance of precursor exhausted, potentially immune checkpoint blockade (ICB) responsive, CD8 T cells. Furthermore, T cell depletion abrogated the survival benefit observed in CaMKK2-/- mice. Considering these T cell phenotypes, we treated CaMKK2-/- mice with ICB, and indeed they were sensitive. To determine if the CaMKK2-/- survival phenotype and ICB response depended on CaMKK2 expression in hematopoietic or in non-hematopoietic cells, we utilized a reciprocal chimera model. Loss of CaMKK2 in the non-hematopoietic cells was more vital for survival and ICB response than in hematopoietic cells, suggesting a potential novel and unique role for CaMKK2 in brain native cells - potentially neurons - in coordinating ICB resistance. CONCLUSIONS We find that CaMKK2 exacerbates mortality and drives ICB resistance by limiting the anti-tumor response in GBM via both hematopoietic and brain native cells. Our findings identify a novel therapeutic target for GBM, and a unique role for CaMKK2 in the TME.

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255 Investigating VISTA’s role intrinsic to T cells in the tumor microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.255 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A276-A276

Author(s):

Cassandra Gilmour ◽

Li Wang ◽

Juan Dong ◽

Sarah Stone ◽

Keman Zhang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Tumor Immunity ◽

Patient Survival ◽

Cytotoxic T Cells ◽

Peripheral Tolerance ◽

Checkpoint Blockade

BackgroundCancer immunotherapies, specifically checkpoint blockade therapies, have demonstrated clinical importance for long term patient survival. One of the major limitations to checkpoint blockade therapies, is the low response rate: ~30% with anti-CTLA4 and anti-PD1 treatment. This may be due to heterogeneity of the patients‘ immune system and the tumor microenvironment including T cell inhibitions. There is a clear need to study this phenomenon and develop additional therapies for long term survival to include a broad range of patients. V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA) is a suppressive protein expressed on many cell types in the tumor microenvironment including cytotoxic T cells. VISTA’s role on T cells has been described as maintaining quiescence and peripheral tolerance in a graft vs host disease model, but is not fully understood in context of the tumor microenvironment.MethodsWe use a series of invivo experiments, including T cell specific VISTA knock outs, to understand the role of VISTA on T cells in the tumor microenvironment.ResultsHere we show a series of in vivo experiments that suggest VISTA has a potent intrinsic role on T cells and therefore anti-tumor immunity. Using a T cell specific VISTA knock out, our results suggest that the absence of VISTA on T cells in combination with anti-CTLA4 and vaccine is a very powerful tumor suppressor compared to vaccine and anti-CTLA4 treatment alone. These results also indicate that the absence of VISTA alters the phenotype of cytotoxic T cells in several ways including the production of inflammatory cytokines.ConclusionsOur preliminary data provides foundation to study VISTA’s role intrinsic to T cells in the tumor microenvironment and how disrupting VISTA’s influence intrinsic to T cells may be advantageous for anti-tumor immunity and long term patient survival.Ethics ApprovalAll in vivo studies were reviewed and approved by Institutional Animal Care and Use Committee (Approval number 2019–2142).

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P03.02 Protein-based cancer vaccine combined with an oncolytic vaccine promotes potent antitumor immunity

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.25 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A13.2-A14

Author(s):

E Belnoue ◽

K Das ◽

M Rossi ◽

T Hofer ◽

S Danklmaier ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Oncolytic Virus ◽

Antitumor Immunity ◽

Checkpoint Blockade ◽

Tumor Models ◽

Tumor Associated Antigens ◽

T Cell Infiltration ◽

Part Time

BackgroundKISIMATM platform allows the development of protein-based cancer vaccines able to induce a potent, tumor-specific CD8 and CD4 T cells response. While the cell penetrating peptide and peptide agonist for Toll like receptor (TLR)-2 and TLR-4 confer, respectively, the cell delivery and self-adjuvanticity properties, the multiantigenic domain allows the targeting of different cancer antigens, resulting in anti-tumoral efficacy in different murine models. Oncolytic viruses exert their therapeutic effects by a prolonged oncolytic action and the associated intratumoral inflammation as well as general immune activation. Arming oncolytic virus with tumor associated antigens can additionally enhance the tumor-specific T cell portion and therefore positively affect the balance of antitumor versus antiviral immune responses. The protein vaccine KISIMATM and the recombinant oncolytic virus VSV-GP-TAA (vesicular stomatitis virus pseudotyped with LCMV GP expressing tumor-associated antigens) are both promising vaccine candidates that offer a new cancer vaccination opportunity when combined in heterologous prime-boost regimen.Materials and MethodsMice were vaccinated with subcutaneous (s.c.) injection of KISIMA-TAA vaccine and/or with intravenous injection of VSV-GP-TAA in different settings. Immunogenicity was assessed by measuring the peripheral antigen-specific response. Anti-tumoral efficacy as well as in depth monitoring of TILs and tumor microenvironment modulation were assessed following therapeutic vaccination in different tumor models. Additionally, transcriptome and immunohistochemistry analyses of the TC-1 tumor have been performed. Combination of heterologous prime-boost with checkpoint blockade PD-1 therapy has been assessed.ResultsPriming with KISIMA-TAA followed by VSV-GP-TAA boost induced a large pool of polyfunctional and persistent antigen-specific cytotoxic T cells in the periphery as well as within the tumor in several tumor models. Frequencies of antigen specific T cells are significantly higher than the respective homologous vaccinations. Additionally, transcriptome analysis of a cold tumor model revealed profound changes in the tumor microenvironment upon heterologous vaccination, including a strong upregulation of gene signatures of several pro-inflammatory cytokines and chemokines required for antitumor immunity along with dendritic and T cell trafficking and activation. This was corroborated by flow-cytometric analysis of tumor-infiltrating leukocytes showing massive CD8+ and CD4+ T cell infiltration as well as repolarization of M2-like macrophages towards M1-phenotype. The presence of the CD8+ T cells within the tumor core was confirmed by immunohistochemistry analysis. Moreover, combining heterologous vaccination with checkpoint blockade further improved its therapeutic efficacy and the number of long-term survivors.ConclusionsThe KISIMA/VSV-GP heterologous prime-boost approach holds great promise for patients with primary or acquired resistance to checkpoint blockade due to its ability to induce tumor-specific T cell, improve T cell infiltration and increase tumor inflammation, even in tumors with limited permissivity for the oncolytic virus.Disclosure InformationE. Belnoue: A. Employment (full or part-time); Significant; AMAL Therapeutics SA. K. Das: None. M. Rossi: A. Employment (full or part-time); Significant; AMAL Therapeutics SA. T. Hofer: None. S. Danklmaier: None. T. Nolden: A. Employment (full or part-time); Significant; Viratherapeutics GmbH. L. Schreiber: None. K. Angerer: None. J. Kimpel: None. S. Hoegler: None. L. Kenner: None. D. von Laer: None. K. Elbers: A. Employment (full or part-time); Significant; Viratherapeutics GmbH. G. Wollmann: None. M. Derouazi: A. Employment (full or part-time); Significant; AMAL Therapeutics SA.

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The CDK4/6 Inhibitor Abemaciclib Induces a T Cell Inflamed Tumor Microenvironment and Enhances the Efficacy of PD-L1 Checkpoint Blockade

Cell Reports ◽

10.1016/j.celrep.2018.02.053 ◽

2018 ◽

Vol 22 (11) ◽

pp. 2978-2994 ◽

Cited By ~ 114

Author(s):

David A. Schaer ◽

Richard P. Beckmann ◽

Jack A. Dempsey ◽

Lysiane Huber ◽

Amelie Forest ◽

...

Keyword(s):

T Cell ◽

Tumor Microenvironment ◽

Checkpoint Blockade

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Association of Tumor Microenvironment T-Cell Repertoire and Mutational Load With Clinical Outcome After Sequential Checkpoint Blockade in Melanoma

immuneACCESS ◽

10.21417/ey2019cir ◽

2019 ◽

Author(s):

E Yusko ◽

M Vignali ◽

RK Wilson ◽

ER Mardis ◽

FS Hodi ◽

...

Keyword(s):

T Cell ◽

Tumor Microenvironment ◽

Clinical Outcome ◽

Checkpoint Blockade ◽

Mutational Load ◽

T Cell Repertoire ◽

Cell Repertoire

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561 Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.561 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A590-A590

Author(s):

Kristin Anderson ◽

Yapeng Su ◽

Madison Burnett ◽

Breanna Bates ◽

Magdalia Rodgers Suarez ◽

...

Keyword(s):

Ovarian Cancer ◽

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Advanced Ovarian Cancer ◽

Checkpoint Blockade ◽

T Cell Therapy ◽

Engineered T Cells ◽

Double Agent ◽

Agent Treatment

BackgroundOver 20,000 women are diagnosed with ovarian cancer annually, and more than half will die within 5 years. This rate has changed little in the last 20 years, highlighting the need for therapy innovation. A promising new strategy with the potential to control tumor growth without toxicity to healthy tissues employs immune T cells engineered to target proteins uniquely overexpressed in tumors. Mesothelin (Msln) contributes to the malignant and invasive phenotype in ovarian cancer, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors.MethodsThe ID8VEGF mouse cell line was used to evaluate if T cells engineered to express a mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill murine ovarian tumor cells in vitro and in vivo. Tumor-bearing mice were treated with TCRMsln T cells plus anti-PD-1, anti-Tim-3 or anti-Lag-3 checkpoint-blocking antibodies administered alone or in combination, ultimately allowing targeting up to three inhibitory receptors simultaneously. Single cell RNA sequencing was used to profile the impact of combination checkpoint blockade on both the engineered T cells and the tumor microenvironment.ResultsIn a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that elements in the tumor microenvironment (TME) limited engineered T cell persistence and ability to kill cancer cells. Triple checkpoint blockade, but not single- or double-agent treatment, dramatically increased anti-tumor function by intratumoral TCRMsln T cells. Single cell RNA-sequencing revealed distinct transcriptome changes in engineered T cells and the TME following triple blockade compared to single- and double-agent treatment. Moreover, combining adoptive immunotherapy with triple checkpoint blockade prolonged survival in the cohort of treated tumor-bearing mice, relative to TCRMsln with or without anti-PD1, or double-agent treatments.ConclusionsInhibitory receptor/ligand interactions within the tumor microenvironment can dramatically reduce T cell function, suggesting tumor cells may evade T cell responses by upregulating the ligands for PD-1, Tim-3 and Lag-3. In a model of advanced ovarian cancer, triple checkpoint blockade significantly improved the function of transferred engineered T cells and improved outcomes in mice in a setting in which single checkpoint blockade had no significant activity. The results suggest that T cell therapy with triple blockade, which can ultimately be more safely pursed in a cell intrinsic form through T cell genetic engineering, may overcome barriers to achieving therapeutic efficacy in patients.Ethics ApprovalThe Institutional Animal Care and Use Committees of the University of Washington and the Fred Hutchinson Cancer Research Center approved all animal studies.

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Chromatin State and Immunotherapy

Blood ◽

10.1182/blood-2018-99-109432 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-16-SCI-16

Author(s):

W. Nicholas Haining

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Microenvironment ◽

Viral Infection ◽

Cd8 T Cells ◽

Research Funding ◽

Checkpoint Blockade ◽

Chronic Viral Infection ◽

T Cell Exhaustion ◽

Cell Exhaustion

Abstract The functional impairment of T cell-mediated immunity within the tumor microenvironment (TME) is a defining feature of many cancers. Checkpoint blockade therapy seeks to reinvigorate T cell responses by targeting inhibitory receptors such as PD-1, which are upregulated by dysfunctional TILs. However, the fundamental mechanisms underlying T cell dysfunction in the TME remain poorly understood, as are the mechanisms by which checkpoint blockade overcomes this dysfunction. Initial studies of dysfunctional CD8+ T cells in both human and mouse tumors suggested that they share features of T cell exhaustion, including co-inhibitory receptor upregulation and defects in cytokine production. However, more recent studies have suggested that TIL dysfunction is a unique state that is distinct from T cell exhaustion. Here we show that anti-PD-1 therapy acts on a specific subpopulation of CD8+ tumor-infiltrating lymphocytes (TILs) in melanoma mouse models as well as patients with melanoma. We find that dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of T cell exhaustion, mirroring those seen in chronic viral infection. Similar to chronic viral infection, exhausted CD8+ TILs contain a subpopulation of "stem-like exhausted" T cells that have a distinct regulatory state. Stem-like exhausted TILs also have critical functional attributes that are not shared by the majority "terminally exhausted" TILs: they retain more polyfunctionality, persist following transfer into tumor-bearing mice, and differentiate to repopulate terminally exhausted TILs in the TME. As a result, stem-like exhausted CD8+ TILs are better able to control tumor growth than terminally exhausted cells. Stem-like exhausted, but not terminally exhausted, CD8+ TILs can respond to anti-PD-1 therapy without reversion of their exhausted epigenetic state. CD8+ T cells with a stem-like exhausted phenotype can be found in human melanoma samples and patients with a higher fraction of this subpopulation in their tumors have a significantly longer duration of response to combination checkpoint blockade therapy. Responsiveness to checkpoint blockade is therefore restricted to a subpopulation of exhausted TILs that retain specific functional properties which enable them to control tumors. Approaches to expand stem-like exhausted CD8+ T cells in the tumor microenvironment may be an important component of improving checkpoint blockade response. Disclosures Haining: Rheos Medicines: Consultancy; Iomx Therapeutics: Consultancy; Third Rock Ventures: Consultancy; Roche: Research Funding; Calico: Research Funding; Novartis: Research Funding; Tango Therapeutics: Consultancy, Equity Ownership.

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