Development of intravenously administered synthetic RNA virus immunotherapy for the treatment of cancer.

The therapeutic effectiveness of oncolytic viruses (OVs) delivered intravenously is limited by the development of neutralizing antibody responses against the virus. To circumvent this limitation and to enable repeated systemic administration of OVs, here we develop Synthetic RNA viruses consisting of a viral RNA genome (vRNA) formulated within lipid nanoparticles. For two Synthetic RNA virus drug candidates, Seneca Valley virus (SVV) and Coxsackievirus A21, we demonstrate vRNA delivery and replication, virus assembly, spread and lysis of tumor cells leading to potent anti-tumor efficacy, even in the presence of OV neutralizing antibodies in the bloodstream. Synthetic-SVV replication in tumors promotes immune cell infiltration, remodeling of the tumor microenvironment, and enhances the activity of anti-PD-1 checkpoint inhibitor. In mouse and non-human primates, Synthetic-SVV is well tolerated reaching exposure well above the requirement for anti-tumor activity. Altogether, the Synthetic RNA virus platform provides an approach that enables repeat intravenous administration of viral immunotherapy.

A Phase I/II Trial of Cetuximab in Combination with Interleukin-12 Administered to Patients with Unresectable Primary or Recurrent Head and Neck Squamous Cell Carcinoma.

PURPOSE: mAbs including cetuximab can induce antibody-dependent cellular cytotoxicity (ADCC) and cytokine production mediated via innate immune cells with the ability to recognize mAb-coated tumors. Preclinical modeling has shown that costimulation of natural killer (NK) cells via the Fc receptor and the IL12 receptor promotes NK-cell-mediated ADCC and production of cytokines. PATIENTS AND METHODS: This phase I/II trial evaluated the combination of cetuximab with IL12 for the treatment of EGFR-expressing head and neck cancer. Treatment consisted of cetuximab 500 mg/m2 i.v. every 2 weeks with either 0.2 mcg/kg or 0.3 mcg/kg IL12 s.c. on days 2 and 5 of the 2-week cycle, beginning with cycle 2. Correlative studies from blood draws obtained prior to treatment and during therapy included measurement of ADCC, serum cytokine, and chemokine analysis, determination of NK cell FcγRIIIa polymorphisms, and an analysis of myeloid-derived suppressor cell (MDSC) frequency in peripheral blood. RESULTS: The combination of cetuximab and IL12 was well tolerated. No clinical responses were observed, however, 48% of patients exhibited prolonged progression-free survival (PFS; average of 6.5 months). Compared with patients that did not exhibit clinical benefit, patients with PFS >100 days exhibited increased ADCC as therapy continued compared with baseline, greater production of IFNγ, IP-10, and TNFα at the beginning of cycle 8 compared with baseline values and had a predominance of monocytic MDSCs versus granulocytic MDSCs prior to therapy. CONCLUSIONS: Further investigation of IL12 as an immunomodulatory agent in combination with cetuximab in head and neck squamous cell carcinoma is warranted.

Immunogenomic correlates of response to cetuximab monotherapy in head and neck squamous cell carcinoma.

BACKGROUND: Mechanisms of resistance to immune-modulating cancer treatments are poorly understood. Using a novel cohort of patients with head and neck squamous cell carcinoma (HNSCC), we investigated mechanisms of immune escape from epidermal growth factor receptor-specific monoclonal antibody (mAb) therapy. METHODS: HNSCC tumors (n = 20) from a prospective trial of neoadjuvant cetuximab monotherapy underwent whole-exome sequencing. Expression of killer-cell immunoglobulin-like receptor (KIR) and human leukocyte antigen-C (HLA-C) and the effect of KIR blockade were assessed in HNSCC cell lines. RESULTS: Nonresponders to cetuximab had an increased rate of mutations in HLA-C compared to responders and HNSCC tumors (n = 528) in The Cancer Genome Atlas (P < 0.00001). In vitro, cetuximab-activated natural killer (NK) cells induced upregulation of HLA-C on HNSCC cells (P < 0.01) via interferon gamma. Treatment of NK cells with the anti-KIR mAb lirilumab increased killing of HNSCC cells (P < 0.001). CONCLUSIONS: Alterations in HLA-C may provide a mechanism of immune evasion through disruption of NK activation.

Novel Effector Phenotype of Tim-3+ Regulatory T Cells Leads to Enhanced Suppressive Function in Head and Neck Cancer Patients.

Purpose: Regulatory T (Treg) cells are important suppressive cells among tumor-infiltrating lymphocytes (TIL). Treg cells express the well-known immune checkpoint receptor PD-1, which is reported to mark "exhausted" Treg with lower suppressive function. T-cell immunoglobulin mucin (Tim)-3, a negative regulator of Th1 immunity, is expressed by a sizeable fraction of TIL Tregs, but the functional status of Tim-3+ Tregs remains unclear.Experimental Design: CD4+CTLA-4+CD25high Treg cells were sorted from freshly excised head and neck squamous cell carcinoma (HNSCC) TIL based on Tim-3 expression. Functional and phenotypic features of these Tim-3+ and Tim-3- TIL Tregs were tested by in vitro suppression assays and multi-color flow cytometry. Gene-expression profiling and NanoString analysis of Tim-3+ TIL Treg were performed. A murine HNSCC tumor model was used to test the effect of anti-PD-1 immunotherapy on Tim-3+ Treg.Results: Despite high PD-1 expression, Tim-3+ TIL Treg displayed a greater capacity to inhibit naïve T-cell proliferation than Tim-3- Treg. Tim-3+ Treg from human HNSCC TIL also displayed an effector-like phenotype, with more robust expression of CTLA-4, PD-1, CD39, and IFN-γ receptor. Exogenous IFN-γ treatment could partially reverse the suppressive function of Tim-3+ TIL Treg. Anti-PD-1 immunotherapy downregulated Tim-3 expression on Tregs isolated from murine HNSCC tumors, and this treatment reversed the suppressive function of HNSCC TIL Tregs.Conclusions: Tim-3+ Treg are functionally and phenotypically distinct in HNSCC TIL, and are highly effective at inhibiting T-cell proliferation despite high PD-1 expression. IFN-γ induced by anti-PD-1 immunotherapy may be beneficial by reversing Tim-3+ Treg suppression. Clin Cancer Res; 24(18); 4529-38. ©2018 AACR.

Co-stimulation of the fc receptor and interleukin-12 receptor on human natural killer cells leads to increased expression of cd25.

Natural killer (NK) cells serve a critical role in the immune response against microbes and developing tumors. We have demonstrated that NK cells produce stimulatory cytokines (e.g., IFN-γ) in response to potent stimulation via immobilized IgG (to engage Fc receptors) and interleukin (IL)-12. CD25 is a component of the high-affinity IL-2R, which promotes NK cell activation in response to low doses of IL-2 such as those released by activated T cells. We hypothesized that stimulation of NK cells via IgG and IL-12 would enhance CD25 expression and promote NK cell anti-tumor activity in response to low-dose IL-2. It was confirmed that this dual stimulation strategy significantly enhanced NK cell CD25 expression compared to unstimulated cells or cells treated with IgG or IL-12 alone. Dual stimulated NK cells also were more responsive to low-dose IL-2. Dual stimulated NK cells subsequently treated with low-dose IL-2 (10 pg/mL) displayed enhanced intracellular signaling as indicated by increased pSTAT5 levels. IFN-γ production and cytotoxicity against K562 cells by NK cells stimulated with low-dose IL-2 was comparable to that of cells treated with high-dose IL-2 (10 ng/mL). Importantly, cells isolated from head and neck cancer patients receiving the mAb cetuximab and IL-12 on a clinical trial displayed increased CD25 expression following combination therapy compared to baseline. Altogether, these findings suggest that FcR and IL-12R co-stimulation induces expression of the high-affinity IL-2R and promotes NK cell anti-tumor activity.

MICA-Expressing Monocytes Enhance Natural Killer Cell Fc Receptor-Mediated Antitumor Functions.

Natural killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNγ, MIP-1α, and TNFα) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as IL12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a Transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA-NKG2D interactions resulted in reductions in IFNγ production. Depletion of monocytes in vivo resulted in decreased IFNγ production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock-depleted controls (P < 0.05). These data suggest that NK cell-monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer. Cancer Immunol Res; 5(9); 778-89. ©2017 AACR.

Activation of the FcgammaReceptorIIIa on human natural killer cells leads to increased expression of functional interleukin-21 receptor.

Natural killer (NK) cells are innate immune effector cells that play a crucial role in immune surveillance and the destruction of cancer cells. NK cells express a low-affinity receptor for the Fc or constant region of immunoglobulin G (FcγRIIIa) and multiple cytokine receptors that respond to antibody-coated targets and cytokines in the tumor microenvironment. In the present work, microarray gene expression analysis revealed that the IL-21 receptor (IL-21R) was strongly upregulated following FcR stimulation. The IL-21R was found to be upregulated on FcR-stimulated NK cells at the transcript level as determined by reverse transcription polymerase chain reaction (RT-PCR). Immunoblot analysis revealed that protein expression of the IL-21R peaked at 8 h post-stimulation of the FcR. Inhibition of the mitogen-activated protein kinase (MAPK) pathway downstream of the FcR blocked the induction of IL-21R expression. Increased expression of the IL-21R sensitized NK cells to IL-21 stimulation, as treatment of FcR-stimulated NK cells led to significantly increased phosphorylation of STAT1 and STAT3, as measured by intracellular flow cytometry and immunoblot analysis. Following FcR-stimulation, IL-21-activated NK cells were better able to mediate the lysis of trastuzumab-coated human epidermal growth factor receptor 2 (HER2+) SK-BR-3 tumor cells as compared to control-treated cells. Likewise, IL-21-induced NK cell secretion of IFNγ following exposure to antibody-coated tumor cells was enhanced following FcR-stimulation. The analysis of NK cells from patients receiving trastuzumab therapy for HER2+ cancer exhibited increased levels of the IL-21R following the administration of antibody suggesting that the presence of monoclonal antibody-coated tumor cells in vivo can stimulate the increased expression of IL-21R on NK cells.

IL-21 Enhances Natural Killer Cell Response to Cetuximab-Coated Pancreatic Tumor Cells.

PURPOSE: Alternative strategies to EGFR blockage by mAbs is necessary to improve the efficacy of therapy in patients with locally advanced or metastatic pancreatic cancer. One such strategy includes the use of NK cells to clear cetuximab-coated tumor cells, as need for novel therapeutic approaches to enhance the efficacy of cetuximab is evident. We show that IL-21 enhances NK cell-mediated effector functions against cetuximab-coated pancreatic tumor cells irrespective of KRAS mutation status. EXPERIMENTAL DESIGN: NK cells from normal donors or donors with pancreatic cancer were used to assess ADCC, IFN-γ release, and T-cell chemotaxis toward human pancreatic cancer cell lines. The in vivo efficacy of IL-21 in combination with cetuximab was evaluated in a subcutaneous and intraperitoneal model of pancreatic cancer. RESULTS: NK cell lysis of cetuximab-coated wild-type and mutant kras pancreatic cancer cell lines were significantly higher following NK cell IL-21 treatment. In response to cetuximab-coated pancreatic tumor cells, IL-21-treated NK cells secreted significantly higher levels of IFN-γ and chemokines, increased chemotaxis of T cells, and enhanced NK cell signal transduction via activation of ERK and STAT1. Treatment of mice bearing subcutaneous or intraperitoneal EGFR-positive pancreatic tumor xenografts with mIL-21 and cetuximab led to significant inhibition of tumor growth, a result further enhanced by the addition of gemcitabine. CONCLUSIONS: These results suggest that cetuximab treatment in combination with IL-21 adjuvant therapy in patients with EGFR-positive pancreatic cancer results in significant NK cell activation, irrespective of KRAS mutation status, and may be a potential therapeutic strategy. Clin Cancer Res; 23(2); 489-502. ©2016 AACR.

Interferon-γ Promotes Antibody-mediated Fratricide of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.

A Phase I Trial to Evaluate Antibody-Dependent Cellular Cytotoxicity of Cetuximab and Lenalidomide in Advanced Colorectal and Head and Neck Cancer.

mAbs can induce antibody-dependent cellular cytotoxicity (ADCC) via the innate immune system's ability to recognize mAb-coated cancer cells and activate immune effector cells. Lenalidomide is an immunomodulatory agent with the capacity to stimulate immune cell cytokine production and ADCC activity. This phase I trial evaluated the combination of cetuximab with lenalidomide for the treatment of advanced colorectal and head and neck squamous cell cancers (HNSCC). This trial included patients with advanced colorectal cancer or HNSCC. Treatment consisted of cetuximab 500 mg/m(2) i.v. every two weeks with lenalidomide given orally days 1-21 on a 28-day cycle. Three dose levels of lenalidomide were evaluated (15, 20, 25 mg). Correlative studies included measurement of ADCC, FcγRIIIA polymorphism genotyping, measurement of serum cytokine levels, and flow cytometric analysis of immune cell subtypes. Twenty-two patients were enrolled (19 colorectal cancer, 3 HNSCC). Fatigue was the only dose-limiting toxicity. One partial response was observed and 8 patients had stable disease at least 12 weeks. The recommended phase II dose is cetuximab 500 mg/m(2) with lenalidomide 25 mg daily, days 1-21. Correlative studies demonstrated a dose-dependent increase in natural killer cytotoxic activity with increasing doses of lenalidomide. Cetuximab and lenalidomide were well tolerated. There was a lenalidomide dose-dependent increase in ADCC with higher activity in patients enrolled in cohort 3 than those enrolled in cohorts 1/2. Although response was not a primary endpoint, there was evidence of antitumor activity for the combination therapy. Further investigation of lenalidomide as an immunomodulator in solid tumors is warranted. Mol Cancer Ther; 15(9); 2244-50. ©2016 AACR.

Folate-conjugated immunoglobulin targets melanoma tumor cells for NK cell effector functions.

The folate receptor (FR) is overexpressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is overexpressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis using KB (human oral epithelial) and F01 (human melanoma) as a positive and a negative control, respectively. FR-positive and FR-negative cell lines were treated with F-IgG or control immunoglobulin G in the presence or absence of cytokines to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells increased following treatment with F-IgG compared with control immunoglobulin G at all effector : target (E : T) ratios (P<0.01). This trend further increased by NK cell stimulation with the activating cytokine interleukin-12. NK cell production of cytokines such as interferon-gamma, macrophage inflammatory protein 1α, and regulated on activation normal T-cell expressed and secreted (RANTES) was also significantly increased in response to costimulation with interleukin-12 stimulation and F-IgG-coated Mel 39 target cells compared with controls (P<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells, which can be further increased by the addition of cytokines.

Determination of the secondary structure of group II bulge loops using the fluorescent probe 2-aminopurine.

Eleven RNA hairpins containing 2-aminopurine (2-AP) in either base-paired or single nucleotide bulge loop positions were optically melted in 1 M NaCl; and, the thermodynamic parameters ΔH°, ΔS°, ΔG°37, and TM for each hairpin were determined. Substitution of 2-AP for an A (adenosine) at a bulge position (where either the 2-AP or A is the bulge) in the stem of a hairpin, does not affect the stability of the hairpin. For group II bulge loops such as AA/U, where there is ambiguity as to which of the A residues is paired with the U, hairpins with 2-AP substituted for either the 5' or 3' position in the hairpin stem have similar stability. Fluorescent melts were performed to monitor the environment of the 2-AP. When the 2-AP was located distal to the hairpin loop on either the 5' or 3' side of the hairpin stem, the change in fluorescent intensity upon heating was indicative of an unpaired nucleotide. A database of phylogenetically determined RNA secondary structures was examined to explore the presence of naturally occurring bulge loops embedded within a hairpin stem. The distribution of bulge loops is discussed and related to the stability of hairpin structures.