Intraperitoneal Monocytes plus IFNs as a Novel Cellular Immunotherapy for Ovarian Cancer: Mechanistic Characterization and Results from a Phase I Clinical Trial.

PURPOSE: Ovarian cancer is the most lethal gynecologic cancer and intrinsically resistant to checkpoint immunotherapies. We sought to augment innate immunity, building on previous work with IFNs and monocytes. PATIENTS AND METHODS: Preclinical experiments were designed to define the mechanisms of cancer cell death mediated by the combination of IFNs α and γ with monocytes. We translated these preclinical findings into a phase I trial of autologous IFN-activated monocytes administered intraperitoneally to platinum-resistant or -refractory ovarian cancer patients. RESULTS: IFN-treated monocytes induced caspase 8-dependent apoptosis by the proapoptotic TRAIL and mediated by the death receptors 4 and 5 (DR4 and DR5, respectively) on cancer cells. Therapy was well tolerated with evidence of clinical activity, as 2 of 9 evaluable patients had a partial response by RECIST criteria, and 1 additional patient had a CA-125 response. Upregulation of monocyte-produced TRAIL and cytokines was confirmed in peripheral blood. Long-term responders had alterations in innate and adaptive immune compartments. CONCLUSIONS: Given the mechanism of cancer cell death, and the acceptable tolerability of the clinical regimen, this platform presents a possibility for future combination therapies to augment anticancer immunity. See related commentary by Chow and Dorigo, p. 299.

Production of a cellular product consisting of monocytes stimulated with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for human use.

BACKGROUND: Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood. METHODS: Counter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction. RESULTS: Both iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. CONCLUSION: Large scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.

Combination immunotherapy with IL-4 Pseudomonas exotoxin and IFN-α and IFN-γ mediate antitumor effects in vitro and in a mouse model of human ovarian cancer.

AIM: We have shown that IL-4 fused to Pseudomonas exotoxin (IL-4-PE) is cytotoxic to ovarian cancer cell lines. The antineoplastic properties of IFN-α, IFN-γ and IL-4-PE have been studied and showed some promise in the clinical trials. Here, we investigated whether the combination of IL-4-PE, IFN-α and IFN-γ will result in increased ovarian cancer cell death in vitro and in vivo. MATERIALS & METHODS: Ovarian cancer cells were tested in vitro to analyze the cytotoxic effects of IL-4-PE, IFN-α and IFN-γ, and the combination of all three. Tumor-bearing xenograft mice were treated with the combination of IL-4-PE, IFN-α and IFN-γ to monitor their overall survival. The JAK/STAT phosphorylation signaling pathways were studied to delineate the mechanism of synergistic antitumor activity. RESULTS: The combination of IL-4-PE with IFN-α and IFN-γ resulted in increased ovarian cancer cell death in vitro and in vivo. Mechanistically, the synergistic antitumor effect was dependent on interferon signaling, but not IL-4-PE signaling as determined by signaling specific chemical inhibitors. The combination therapy induced the activation of critical mediators of apoptosis. CONCLUSION: The combination of IL-4-PE with interferons increased overall survival of mice with human ovarian cancer xenograft. These data suggest that this novel combination could provide a unique approach to treating ovarian cancer.

Human macrophages survive and adopt activated genotypes in living zebrafish.

The inflammatory response, modulated both by tissue resident macrophages and recruited monocytes from peripheral blood, plays a critical role in human diseases such as cancer and neurodegenerative disorders. Here, we sought a model to interrogate human immune behavior in vivo. We determined that primary human monocytes and macrophages survive in zebrafish for up to two weeks. Flow cytometry revealed that human monocytes cultured at the physiological temperature of the zebrafish survive and differentiate comparable to cohorts cultured at human physiological temperature. Moreover, key genes that encode for proteins that play a role in tissue remodeling were also expressed. Human cells migrated within multiple tissues at speeds comparable to zebrafish macrophages. Analysis of gene expression of in vivo educated human macrophages confirmed expression of activated macrophage phenotypes. Here, human cells adopted phenotypes relevant to cancer progression, suggesting that we can define the real time immune modulation of human tumor cells during the establishment of a metastatic lesion in zebrafish.

A Phase 1 trial of autologous monocytes stimulated ex vivo with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for intra-peritoneal administration in recurrent ovarian cancer.

BACKGROUND: Ovarian cancer has no definitive second line therapeutic options, and largely recurs in the peritoneal cavity. Locoregional immune therapy using both interferons and monocytes can be used as a novel approach. Interferons have both cytostatic and cytotoxic properties, while monocytes stimulated with interferons have potent cytotoxic properties. Due to the highly immune suppressive properties of ovarian cancer, ex vivo stimulation of autologous patient monocytes with interferons and infusion of all three agents intraperitoneally (IP) can provide a strong pro-inflammatory environment at the site of disease to kill malignant cells. METHODS: Patient monocytes are isolated through counterflow elutriation and stimulated ex vivo with interferons and infused IP through a semi-permanent catheter. We have designed a standard 3 + 3 dose escalation study to explore the highest tolerated dose of interferons and monocytes infused IP in patients with chemotherapy resistant ovarian cancer. Secondary outcome measurements of changes in the peripheral blood immune compartment and plasma cytokines will be studied for correlations of response. DISCUSSION: We have developed a novel immunotherapy focused on the innate immune system for the treatment of ovarian cancer. We have combined the use of autologous monocytes and interferons alpha and gamma for local-regional administration directly into the peritoneal cavity. This therapy is highly unique in that it is the first study of its type using only components of the innate immune system for the locoregional delivery consisting of autologous monocytes and dual interferons alpha and gamma. Trial Registration ClinicalTrials.gov Identifier: NCT02948426, registered on October 28, 2016. https://clinicaltrials.gov/ct2/show/NCT02948426.

Current prospects of type II interferon γ signaling and autoimmunity.

Interferon γ (IFNγ) is a pleiotropic protein secreted by immune cells. IFNγ signals through the IFNγ receptor, a protein complex that mediates downstream signaling events. Studies into IFNγ signaling have provided insight into the general concepts of receptor signaling, receptor internalization, regulation of distinct signaling pathways, and transcriptional regulation. Although IFNγ is the central mediator of the adaptive immune response to pathogens, it has been shown to be involved in several non-infectious physiological processes. This review will provide an introduction into IFNγ signaling biology and the functional roles of IFNγ in the autoimmune response.

Monocyte and interferon based therapy for the treatment of ovarian cancer.

Cytokines and cells of the innate immune system have been shown to be critical regulators in the elimination, equilibrium and escape of malignant cells. Despite in vitro and in vivo evidence, components of the innate immune system have shown limited efficacy in the treatment of ovarian cancer. Intraperitoneal immunotherapies are a promising field that has not yet been fully explored in ovarian cancer. Cytokine immunotherapy using interferon alpha (IFN-α) and interferon gamma (IFN-γ) has predominantly been used intraperitoneally in ovarian cancer, with promising results. Early studies also showed that autologous monocytes infused into the peritoneum have anti-tumor properties. Combination therapies have been shown to be more effective in treating cancer than mono-therapies. Based on these observations the combination of cell therapy with cytokine therapy may provide a unique strategy for the treatment of chemotherapy resistant solid cancers.

Human monocytes in the presence of interferons alpha2a and gamma are potent killers of serous ovarian cancer cell lines in combination with paclitaxel and carboplatin.

Interferons (IFNs) play an important role in immune surveillance of tumors; however, their efficacy in the treatment of malignancies has been limited. Monocytes are mononuclear phagocytes that are critical to the generation of an innate immune response to tumors. The authors and others have shown that treatment of tumor cell lines in vitro and in vivo with human monocytes primed with type I and type II IFNs results in killing. We now expand on this work, in an extended panel of ovarian cancer cell lines. In this study, we hypothesized that there would be variable sensitivity amongst cell lines to the killing properties of monocytes and IFNs. To this end, we explored the interactions of IFN primed monocytes in conjunction with the standard of therapy for ovarian cancer, taxane, and platinum-based chemotherapeutics. Using 6 ovarian cancer cell lines, we demonstrated that there is variation from cell line to cell line in the ability of IFN-α2a and IFN-γ primed monocytes to synergistically kill target tumor cells, and further, there is an additive killing effect when target cells are treated with both IFN primed monocytes and chemotherapy.

A cell permeable peptide targeting the intracellular loop 2 of endothelin B receptor reduces pulmonary hypertension in a hypoxic rat model.

Cell permeable peptides (CPP) aid cellular uptake of targeted cargo across the hydrophobic plasma membrane. CPP-mediated cargo delivery of receptor signaling motifs provides an opportunity to regulate specific receptor initiated signaling cascades. Both endothelin-1 receptors, ETA and ETB, have been targets of antagonist therapies for individuals with pulmonary arterial hypertension (PAH). These therapies have had success but have been accompanied by adverse reactions. Also, unlike the CPP which target specific signaling cascades, the antagonists target the entire function of the receptor. Using the CPP strategy of biased antagonism of the ETB receptor's intracellular loop 2 (ICB2), we demonstrate blunting of hypoxic pulmonary hypertension (HPH) in the rat, including indices of pulmonary arterial pressure, right ventricular hypertrophy and pulmonary vascular remodeling. Further, ex vivo analysis of the pulmonary artery treated with the IC2B peptide upon injection manifests marked reductions in Akt and ERK activation. Both kinases have been intimately related to cell proliferation and vascular contraction, the hallmarks of PAH. These observations in sum illustrate an involvement of the ETB receptor in HPH and furthermore provide a basis for a novel, CPP-based, strategy in the treatment of PAH, ultimately able to target not only ET-1, but also other factors involved in the development of PAH.

Mannose-capped lipoarabinomannan from Mycobacterium tuberculosis preferentially inhibits sphingosine-1-phosphate-induced migration of Th1 cells.

Chemokine receptor cross-desensitization provides an important mechanism to regulate immune cell recruitment at sites of inflammation. We previously reported that the mycobacterial cell wall glycophospholipid mannose-capped lipoarabinomannan (ManLAM) could induce human peripheral blood T cell chemotaxis. Therefore, we examined the ability of ManLAM to desensitize T cells to other chemoattractants as a potential mechanism for impaired T cell homing and delayed lung recruitment during mycobacterial infection. We found that ManLAM pretreatment inhibited in vitro migration of naive human or mouse T cells to the lymph node egress signal sphingosine-1-phosphate (S1P). Intratracheal administration of ManLAM in mice resulted in significant increases in T cells, primarily CCR5(+) (Th1) cells, in lung-draining lymph nodes. To investigate the selective CCR5 effect, mouse T cells were differentiated into Th1 or Th2 populations in vitro, and their ability to migrate to S1P with or without ManLAM pretreatment was analyzed. ManLAM pretreatment of Th1 populations inhibited S1P-induced migration but had no effect on Th2 cell S1P-directed migration, suggesting a differential effect by S1P on the two subsets. The PI3K/AKT inhibitor Ly294002 inhibited S1P-directed migration by Th1 cells, whereas the ERK inhibitor U0126 inhibited Th2 cell S1P-directed migration. These observations demonstrate that S1P-induced migratory responses in Th1 and Th2 lymphocytes occurs via different signaling pathways and suggests further that the production of ManLAM during Mycobacterium tuberculosis infection may function to sequester Th1 cells in lung-draining lymph nodes, thereby delaying their recruitment to the lung.

Enhancing and limiting endothelin-1 signaling with a cell-penetrating peptide mimicking the third intracellular loop of the ETB receptor.

TAT (a 13-mer derived from the HIV-1 Tat protein)-linked cell-permeable peptides deliver plasma membrane impermeable cargos into the cell. We investigated the effect of a TAT-linked intracellular third loop of the endothelin-1 type B receptor on endothelin-1 activation of ERK. The effect of this peptide on ERK activation was determined in ETB receptor cDNA-transfected Chinese hamster ovary cells and in ETA- and ETB-expressing human pulmonary artery smooth muscle cells obtained from a normal and a bone morphogenetic protein-2 receptor, exon 1-8 deletion subject, with pulmonary hypertension. In the Chinese hamster ovary cells the peptide, at optimum 10 µm concentration, suppressed endothelin-1 activation. In the normal human pulmonary artery smooth muscle cells, the peptide marginally enhanced endothelin-1 activation of ERK. However, it markedly enhanced the endothelin-1 activation of ERK in the bone morphogenetic protein-2 receptor human pulmonary artery smooth muscle cells. While the effective concentration for endothelin-1 activation of ERK remained unchanged in the bone morphogenetic protein-2 receptor human pulmonary artery smooth muscle cells, the number of ETB receptors declined by 2/3. These data point to the intracellular third loop peptide as having variable receptor interactive effects with both signal repressive and enhancing capabilities. Peptides that can alter endothelin-1 signal capabilities are potentially important in the study and treatment of pulmonary hypertension.

Human immunodeficiency virus type 1 gp120 reprogramming of CD4+ T-cell migration provides a mechanism for lymphadenopathy.

Infection by human immunodeficiency virus type 1 (HIV-1) is associated with decreases in peripheral CD4(+) T cells and development of lymphadenopathy. The precise mechanisms by which HIV-1 induces these changes have not been elucidated. T-cell trafficking through lymphoid tissues is facilitated by CCL21-mediated entry and sphingosine-1-phosphate (S1P)-mediated egress. Having previously determined that HIV-1 envelop glycoprotein, gp120, directly alters T-cell migration, we investigated whether gp120 without HIV-1 infection could influence the responses of CD4(+) T cells to the signals involved in T-cell trafficking through lymph tissue. Incubation of normal human T cells with gp120 for 1 h resulted in reprogramming of CD4 T-cell migratory responses by increasing sensitivity to CCL20 and CCL21 and complete inhibition of migration to S1P. Incubation of human T cells with gp120 prior to injection into NOD.CB17-Prkdc(scid)/J mice resulted in increases in lymph node accumulation of CD4(+) T cells, with reciprocal decreases in blood and spleen compared to T cells not exposed to gp120. The effects of gp120 required CD4 signaling mediated through p56(lck). These findings suggest that gp120 alone can alter CD4(+) influx and efflux from lymph nodes in a fashion consistent with the development of lymphopenia and lymphadenopathy.

Histamine 4 receptor activation induces recruitment of FoxP3+ T cells and inhibits allergic asthma in a murine model.

Histamine has an important role in regulation of immune response which is mediated by differential expression of four distinct receptors, H1R-H4R. H1R and HR2 have previously been shown to be involved with modulation of lung inflammation. H4R is also expressed on inflammatory cells; therefore, we investigated the potential role of H4R in development of allergic asthma in a murine model. We determined that the H4R agonist 4-methylhistamine when delivered intratracheally before Ag challenge mitigated airway hyperreactivity and inflammation. This was associated with an increase in IL-10 and IFN-gamma, but not TGF-beta or IL-16, as well as a decrease in IL-13 in the bronchoalveolar lavage fluid. We also observed that H4R agonist instillation resulted in accumulation of FoxP3(+) T cells suggesting a direct effect on T regulatory cell recruitment. To investigate this further, we determined the in vitro effect of H4R stimulation on human T cell migration. The H4R agonist induced a 2- to 3-fold increase in T cell migration, similar to that seen for H1R agonists. Cells transmigrating to the H4R agonist, but not H1R, were skewed toward a CD4 cell expressing CD25 and intracellular FoxP3. H4R-responsive cells suppressed proliferation of autologous T cells, an effect that was dependent on IL-10 production. We conclude that H4R stimulation enriches for a regulatory T cell with potent suppressive activity for proliferation. These findings identify a novel function for H4R and suggest a potential therapeutic approach to attenuation of asthmatic inflammation.

IgG-derived Fc down-regulates virus-induced plasmacytoid dendritic cell (pDC) IFNalpha production.

Interferon alpha (IFNalpha) produced primarily by plasmacytoid dendritic cells (pDC) is a potent component of the anti-viral innate immune response, and modulates adaptive immunity. Primary control of IFNalpha production occurs at a cellular level and is highly dependent upon regulatory factors and their products. Recent studies have identified up-regulation of IFNalpha production mediated by the adaptive immune response in the form of immune specific IgG. These studies establish a role for the external control of IFNalpha production. The current work demonstrates that the Fc portion of IgG is a potent inhibitor of IFNalpha produced by pDCs in response to HIV, HSV, and VSV. Fc down-regulation occurs after IFNalpha production can be detected by bioassay, and suggests the existence of late regulatory events in the control of IFNalpha production. Down-regulation of IFNalpha is not caused by Fc-induced necrosis, apoptosis or neutralization of IFNalpha activity. Demonstration of Fc-mediated down-regulation of IFNalpha provides additional evidence of the role of IgG in the regulation of IFNalpha production.

Immune-specific immunoglobulin G-mediated enhancement of human immunodeficiency virus-induced IFN-alpha production.

Interferon-alpha (IFN-alpha) is synthesized as an integral part of innate immunity to viral infection. We previously provided preliminary evidence that antibody-containing serum from HIV-infected individuals enhanced HIV-induced production of IFN-alpha. Subsequently, preparations of pooled human immunoglobulin G (IgG) have also been shown to enhance poliovirus (PV)-induced IFN-alpha production. The current work establishes IgG as the serum mediator that enhances induction of IFN-alpha by HIV. Our studies also establish the ability of sera from individual subjects to enhance PV-induced IFN-alpha production. HIV-induced IFN-alpha production was enhanced maximally by >4000-fold and by an average of 25-fold. Sera from 74 people enhanced PV- induced IFN-alpha from undetectable levels to an average of 615 units (range 7-4679 units). The ability of individual sera to enhance IFN-alpha production by HIV and PV persisted undiminished in patients with AIDS. IgG-mediated enhancement of IFN-alpha production was similar to that induced by IgG and PV and was blocked by IgG Fc fragments. Demonstration of the selective enhancement of HIV-induced IFN-alpha production by IgG from HIV-seropositive individuals provides further evidence for the existence of antigen-specific upregulation of a critical component of innate antiviral immunity by the adaptive Th2 immune response.