Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade

Cancer immunotherapy using antibodies that target immune checkpoints has delivered outstanding results. However, responses only occur in a subset of patients, and it is not fully understood what biological processes determine an effective outcome. This lack of understanding hinders the development of rational combination treatments. We set out to define the pretreatment microenvironment associated with an effective outcome by using the fact that inbred mouse strains bearing monoclonal cancer cell line–derived tumors respond in a dichotomous manner to immune checkpoint blockade (ICB). We compared the cellular composition and gene expression profiles of responsive and nonresponsive tumors from mice before ICB and validated the findings in cohorts of patients with cancer treated with ICB antibodies. We found that responsive tumors were characterized by an inflammatory gene expression signature consistent with up-regulation of signal transducer and activator of transcription 1 (STAT1) and Toll-like receptor 3 (TLR3) signaling and down-regulation of interleukin-10 (IL-10) signaling. In addition, responsive tumors had more infiltrating-activated natural killer (NK) cells, which were necessary for response. Pretreatment of mice with large established tumors using the STAT1-activating cytokine interferon-γ (IFNγ), the TLR3 ligand poly(I:C), and an anti–IL-10 antibody sensitized tumors to ICB by attracting IFNγ-producing NK cells into the tumor, resulting in increased cure rates. Our results identify a pretreatment tumor microenvironment that predicts response to ICB, which can be therapeutically attained. These data suggest a biomarker-driven approach to patient management to establish whether a patient would benefit from treatment with sensitizing therapeutics before ICB.

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P06.12 Combination therapy of CAR-NK-cells and anti-PD-1 antibody results in high efficacy against advanced-stage glioblastoma in a syngeneic mouse model and induces protective anti-tumor immunity in vivo

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.91 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A46.2-A47

Author(s):

F Strassheimer ◽

MI Strecker ◽

T Alekseeva ◽

J Macas ◽

MC Demes ◽

...

Keyword(s):

Combination Therapy ◽

Nk Cells ◽

Tumor Cells ◽

Combined Treatment ◽

Checkpoint Blockade ◽

Great Promise ◽

Advanced Stage ◽

Lymphocyte Infiltration ◽

Term Survival ◽

Syngeneic Mouse Model

BackgroundCheckpoint inhibitors as well as adoptive cell therapy hold great promise for cancer therapy and encouraging treatment responses have already been demonstrated in different cancer indications. Glioblastoma (GB) is the most common and aggressive primary brain tumor. Standard therapy has very limited efficacy in the majority of patients. Analysis of the GB tumor microenvironment (TME) has shown prominent immunosuppressive features including expression of PD-L1 on tumor cells and increased frequency of FOX-P3 positive regulatory T cells. While the surrounding brain is HER2-negative, GB tumors are frequently HER2-positive, suggesting HER2 as a promising target for adoptive immunotherapy. Previous results from mouse glioma models showed efficacy of CAR-NK cells (NK-92/5.28.z) targeted against HER2 as monotherapy with relatively small tumors, but not with advanced late-stage tumors.Materials and MethodsThe murine glioma cell line GL261 was transfected with HER2. Tumor cells were implanted either subcutaneously or orthotopically into C57BL/6 mice and treated either with HER2-specific NK-92/5.28.z cells alone or in combination with an anti-PD-1 antibody. Effects on tumor growth and survival were determined. Lymphocyte infiltration and immunosuppressive TME were characterized in high-dimensional high-throughput analysis via RNAseq and multiplex IHC.ResultsCombined treatment with NK-92/5.28.z cells and anti-PD-1 checkpoint blockade resulted in synergistic effects with tumor regression and long-term survival even of advanced-stage tumor bearing mice. Analysis of TME showed enhanced cytotoxic lymphocyte infiltration and altered profiles of exhaustion markers in tumor and immune cells, leading to an altered TME after combined treatment with NK-92/5.28.z cells and anti-PD-1 antibody.ConclusionsThese data demonstrate that efficacy of NK-92/5.28.z cells can be enhanced in combination with checkpoint blockade, resulting in successful treatment of advanced tumors refractory to NK-92/5.28.z monotherapy. Furthermore, the combination therapy induces a cytotoxic rather than immunosuppressive TME, leading to a primed immune system. To address this question in a clinical setting, we are preparing a combination therapy cohort as part of our ongoing phase I clinical study (CAR2BRAIN; NCT03383978).Disclosure InformationF. Strassheimer: None. M.I. Strecker: None. T. Alekseeva: None. J. Macas: None. M.C. Demes: None. I.C. Mildenberger: None. T. Tonn: None. P.J. Wild: None. L. Sevenich: None. Y. Reiss: None. P.N. Harter: None. K.H. Plate: None. W.S. Wels: None. J.P. Steinbach: None. M.C. Burger: None.

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PD-1 checkpoint blockade in advanced melanoma patients: NK cells, monocytic subsets and host PD-L1 expression as predictive biomarker candidates

OncoImmunology ◽

10.1080/2162402x.2020.1786888 ◽

2020 ◽

Vol 9 (1) ◽

pp. 1786888 ◽

Cited By ~ 1

Author(s):

Yago Pico de Coaña ◽

Maria Wolodarski ◽

Irene van der Haar Àvila ◽

Takahiro Nakajima ◽

Stamatina Rentouli ◽

...

Keyword(s):

Nk Cells ◽

Predictive Biomarker ◽

Advanced Melanoma ◽

Checkpoint Blockade ◽

Melanoma Patients

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Cytokine therapy restores antitumor responses of NK cells rendered anergic in MHC I-deficient tumors

OncoImmunology ◽

10.1080/2162402x.2014.1002725 ◽

2015 ◽

Vol 5 (1) ◽

pp. e1002725 ◽

Cited By ~ 5

Author(s):

Michele Ardolino ◽

David H Raulet

Keyword(s):

Nk Cells ◽

Cytokine Therapy ◽

Mhc I

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Antagonistic anti-LILRB1 monoclonal antibody regulates antitumor functions of natural killer cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2019-000515 ◽

2020 ◽

Vol 8 (2) ◽

pp. e000515 ◽

Cited By ~ 2

Author(s):

Heyu Chen ◽

Yuanzhi Chen ◽

Mi Deng ◽

Samuel John ◽

Xun Gui ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Natural Killer ◽

Xenograft Model ◽

Checkpoint Blockade ◽

Solid Cancer ◽

Tumoricidal Activity ◽

Healthy Donors

BackgroundCurrent immune checkpoint blockade strategies have been successful in treating certain types of solid cancer. However, checkpoint blockade monotherapies have not been successful against most hematological malignancies including multiple myeloma and leukemia. There is an urgent need to identify new targets for development of cancer immunotherapy. LILRB1, an immunoreceptor tyrosine-based inhibitory motif-containing receptor, is widely expressed on human immune cells, including B cells, monocytes and macrophages, dendritic cells and subsets of natural killer (NK) cells and T cells. The ligands of LILRB1, such as major histocompatibility complex (MHC) class I molecules, activate LILRB1 and transduce a suppressive signal, which inhibits the immune responses. However, it is not clear whether LILRB1 blockade can be effectively used for cancer treatment.MethodsFirst, we measured the LILRB1 expression on NK cells from cancer patients to determine whether LILRB1 upregulated on NK cells from patients with cancer, compared with NK cells from healthy donors. Then, we developed specific antagonistic anti-LILRB1 monoclonal antibodies and studied the effects of LILRB1 blockade on the antitumor immune function of NK cells, especially in multiple myeloma models, in vitro and in vivo xenograft model using non-obese diabetic (NOD)-SCID interleukin-2Rγ-null mice.ResultsWe demonstrate that percentage of LILRB1+ NK cells is significantly higher in patients with persistent multiple myeloma after treatment than that in healthy donors. Further, the percentage of LILRB1+ NK cells is also significantly higher in patients with late-stage prostate cancer than that in healthy donors. Significantly, we showed that LILRB1 blockade by our antagonistic LILRB1 antibody increased the tumoricidal activity of NK cells against several types of cancer cells, including multiple myeloma, leukemia, lymphoma and solid tumors, in vitro and in vivo.ConclusionsOur results indicate that blocking LILRB1 signaling on immune effector cells such as NK cells may represent a novel strategy for the development of anticancer immunotherapy.

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Radiotherapy Combined with PD-1 Inhibition Increases NK Cell Cytotoxicity towards Nasopharyngeal Carcinoma Cells

Cells ◽

10.3390/cells10092458 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2458

Author(s):

Anna Makowska ◽

Nora Lelabi ◽

Christina Nothbaum ◽

Lian Shen ◽

Pierre Busson ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Nk Cells ◽

Nk Cell ◽

Immune Checkpoint Blockade ◽

Carcinoma Cells ◽

Checkpoint Blockade ◽

Therapeutic Modality ◽

Release Assay ◽

Nk Cell Cytotoxicity ◽

Calcein Release

Background: Nasopharyngeal carcinoma (NPC) in endemic regions and younger patients is characterized by a prominent lymphomononuclear infiltration. Radiation is the principal therapeutic modality for patients with NPC. Recent data suggest that the efficacy of radiotherapy in various cancers can be augmented when combined with immune checkpoint blockade. Here, we investigate the effect of radiotherapy on the killing of NPC cells by Natural Killer (NK) cells. Methods: NPC cell lines and a patient-derived xenograft were exposed to NK cells in the context of radiotherapy. Cytotoxicity was measured using the calcein-release assay. The contribution of the PD-L1/PD-1 checkpoint and signaling pathways to killing were analyzed using specific inhibitors. Results: Radiotherapy sensitized NPC cells to NK cell killing and upregulated expression of PD-1 ligand (PD-L1) in NPC cells and PD-1 receptor (PD-1) in NK cells. Blocking of the PD-L1/PD-1 checkpoint further increased the killing of NPC cells by NK cells in the context of radiotherapy. Conclusion: Radiation boosts the killing of NPC cells by NK cells. Killing can be further augmented by blockade of the PD-L1/PD-1 checkpoint. The combination of radiotherapy with PD-L1/PD-1 checkpoint blockade could therefore increase the efficacy of radiotherapy in NPC tumors.

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