Activation of effector immune cells promotes tumor stochastic extinction: A homotopy analysis approach

Development of cell-based assay for predictively evaluating the FcγR-mediated human immune cell activation by therapeutic monoclonal antibodies

Cellular metabolism of both cancer and immune cells in the acidic, hypoxic, and nutrient-depleted tumor microenvironment (TME) has attracted increasing attention in recent years. Accumulating evidence has shown that cancer cells in TME could outcompete immune cells for nutrients and at the same time, producing inhibitory products that suppress immune effector cell functions. Recent progress revealed that metabolites in the TME could dysregulate gene expression patterns in the differentiation, proliferation, and activation of immune effector cells by interfering with the epigenetic programs and signal transduction networks. Nevertheless, encouraging studies indicated that metabolic plasticity and heterogeneity between cancer and immune effector cells could provide us the opportunity to discover and target the metabolic vulnerabilities of cancer cells while potentiating the anti-tumor functions of immune effector cells. In this review, we will discuss the metabolic impacts on the immune effector cells in TME and explore the therapeutic opportunities for metabolically enhanced immunotherapy.

The activation of effector immune cells at the cervicovaginal mucosa (CVM) might influence the cervical HIV load and thus the secondary transmission; however, limited information is available about the innate effector cells at CVM during HIV infection. In this study, we quantified and assessed the activation of the effector immune cells at the CVM of HIV-infected women with different disease outcomes: nonprogressive HIV disease (LTNPs) and chronic HIV-infected (CHI) and their relationship with cervical viral shedding. The phenotype and frequency of cytobrush-derived effector immune cells like natural killer cells, T cells, and dendritic cells and their degranulation status (CD107a expression as a surrogate marker of activation) was determined using flow cytometry in age-matched HIV- infected and uninfected women and their association with cervical HIV load was determined. The frequencies of dendritic cells, CD56, CD56 natural killer cell subsets were similar in both the study groups and also within the HIV-infected women with and without progressive disease. The frequencies of CD56CD16 natural killer cells (P = 0.04) and degranulating CD56 natural killer cells were significantly higher among HIV-infected women (P < 0.05). Among HIV-infected women, LTNP women showed reduced degranulation of natural killer and CD8 T cells than seen in the CHI women, which was also associated with lower cervical viral load (P < 0.05). The present study showed that increased degranulation of natural killer and T cells is associated with higher HIV shedding at the CVM of HIV-infected women. Hence reduction of the local immune activation at CVM could be an effective strategy to reduce the cervical viral load.

Ex Vivo Expansion and Activation of Cord Blood Antigen Presenting and Immune Effector Cells

ASTCT Consensus Grading for Cytokine Release Syndrome and Neurologic Toxicity Associated with Immune Effector Cells

This study demonstrates that the combination of Smac mimetic and oncolytic virotherapy (OVT) induces cell death in acute myeloid leukemia (AML) cell lines and primary patient samples; this is the first time the efficacy of this combination approach has been investigated in a human cancer model and for the treatment of AML. Methods: The Smac mimetic LCL161 and several oncolytic viruses (OVs) were tested for in vitro cytotoxicity against a panel of diverse AML cell lines. Bystander killing of LCL161-treated AML cells was explored using conditioned media from OV-treated PBMCs. Multiplex immunoassays were used to detect potential mediators of bystander cytotoxicity produced following OV treatment. Several upregulated cytokines were tested for their ability to enhance LCL161 toxicity in AML cell lines by MTS assay. The activation of healthy donor peripheral blood mononuclear cells (PBMC) was investigated by flow cytometry and immune cell-mediated death of AML cells was measured using chromium release assays. In vitro direct and bystander toxicity was further verified using fresh blood samples from AML patients. Results: A rhabdovirus, MG1, showed the greatest cytotoxicity across a panel of AML cell lines compared to several other OVs. In resistant cell lines the addition of LCL161 treatment led to increased cell death, indicating the potential merits of a combination strategy. Primary AML samples exposed to both LCL161 and MG1 showed greater levels of cell death than either treatment alone, confirming combinational efficacy. Treating healthy donor PBMCs with virus induced the activation of innate immune effector cells, as indicated by an increase in CD69 on NK cells as well as stimulating CD14+ monocytes to produce membrane-bound TNFα-related apoptosis-inducing ligand (TRAIL). Immune cell-mediated death was increased following activation by MG1, and LCL161 sensitized AML cell lines to death by the membrane mimicking KillerTRAIL. Conditioned media from MG1-treated healthy and patient-derived PBMCs displayed an inflammatory milieu that was toxic to AML cell lines. Further analysis identified several cytokines relevant to AML therapy, including the anti-viral interferon alpha (IFNα), soluble TRAIL, and tumor necrosis factor alpha (TNFα); LCL161 was able to increase the sensitivity of AML cells to MG1-conditioned media and recombinant versions of IFNα and TNFα. Discussion: OVT has shown positive clinical efficacy in many solid tumors; however, it is underexplored in the context of hematologic malignancies. Many cases of spontaneous remission following viral infection, the correlation of cytomegalovirus reactivation and positive prognosis following hematologic stem cell transplant, along with the identification of OVs which target leukemic cells without harming healthy hematologic stem cells, suggest that AML patients may be suitable for OVT. Indeed, data from our lab suggest that PBMCs from AML patients are able to recognize and respond to OV, to induce antitumor cytotoxicity. MG1 is cytotoxic to AML cells and acts as an immunogenic agent to induce the release of soluble inflammatory mediators by PBMCs that lead to the death of bystander cells without the need for direct infection. Recent studies into the use of Smac mimetics in hematologic malignancies have demonstrated the synergistic action of combining treatment with inflammatory cytokines, suggesting that combination with an immunogenic agent may be beneficial. Indeed, LCL161 is able to sensitize both AML cell lines and patient-derived AML blasts to MG1 treatment. The release of inflammatory mediators following MG1 treatment also stimulates the activation of innate immune effector cells to induce AML cell death, indicating the ability to initiate a cell-mediated innate immune attack on AML cells using this regime. In conclusion, these data suggest that MG1 should be considered in combination with the Smac mimetic, LCL161, for the treatment of AML. Citation Format: Joanne L. Hopper, Louise ME Müller, Victoria A. Jennings, Gina B. Scott, Stewart McConnell, Richard Kelly, Fiona Errington-Mais. Oncolytic virotherapy enhances Smac mimetic treatment of acute myeloid leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 20.

Most anti-cancer modalities are designed to directly kill cancer cells deploying mechanisms of action (MOAs) centered on the presence of a precise target on cancer cells. The efficacy of these approaches is limited because the rapidly evolving genetics of neoplasia swiftly circumvents the MOA generating therapy-resistant cancer cell clones. Other modalities engage endogenous anti-cancer mechanisms by activating the multi-cellular network (MCN) surrounding neoplastic cells in the tumor microenvironment (TME). These modalities hold a better chance of success because they activate numerous types of immune effector cells that deploy distinct cytotoxic MOAs. This in turn decreases the chance of developing treatment-resistance. Engagement of the MCN can be attained through activation of immune effector cells that in turn kill cancer cells or when direct cancer killing is complemented by the production of proinflammatory factors that secondarily recruit and activate immune effector cells. For instance, adoptive cell therapy (ACT) supplements cancer cell killing with the release of homeostatic and pro-inflammatory cytokines by the immune cells and damage associated molecular patterns (DAMPs) by dying cancer cells. The latter phenomenon, referred to as immunogenic cell death (ICD), results in an exponential escalation of anti-cancer MOAs at the tumor site. Other approaches can also induce exponential cancer killing by engaging the MCN of the TME through the release of DAMPs and additional pro-inflammatory factors by dying cancer cells. In this commentary, we will review the basic principles that support emerging paradigms likely to significantly improve the efficacy of anti-cancer therapy.

ABC transporters were identified originally for their contribution to clinical MDR as a result of their capacity to extrude various unrelated cytotoxic drugs. More recent reports have shown that ABC transporters can play important roles in the development, differentiation, and maturation of immune cells and are involved in migration of immune effector cells to sites of inflammation. Many of the currently identified, endogenous ABC transporter substrates have immunostimulating effects. Increasing the expression of ABC transporters on immune cells and thereby enhancing immune cell development or functionality may be beneficial to immunotherapy in the field of oncology. On the contrary, in the treatment of autoimmune diseases, blockade of these transporters may prove beneficial, as it could dampen disease activity by compromising immune effector cell functions. This review will focus on the expression, regulation, and substrate specificity of ABC transporters in relation to functional activities of immune effector cells and discusses implications for the treatment of cancer on the one hand and autoimmune diseases on the other.

e13031 Background: The anti-tumor activity of the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3) relies on the activation of immune effector cells against EpCAM+ tumor cells. It has been reported for some antibody-based therapies that ethnic groups may differ with respect to responsiveness of immune effector cells. The present nonclinical study therefore investigated potential inter-ethnic differences (Caucasian vs. Asian Donors) with regard to the potency of catumaxomab in vitro. Methods: Blood samples of healthy subjects of different ethnic backgrounds (25 Caucasian, 25 Korean and 15 Japanese donors) were collected for the comparative analysis. PBMCs of the respective blood sample were purified and used as effector cells evaluating catumaxomab-mediated cytotoxicity towards EpCAM+ tumor target cells (HCT-8; colon) in vitro. Results: The comparative analysis of potency obtained for the individual ethnic subgroups (Caucasian, Korean and Japanese) did not reveal major inter-ethnic differences. Even though a wider variability was observed among Caucasians, the potency range observed with immune effector cells of Asian donor origin (Japanese or Korean) was found to be within the potency range observed for the Caucasian ethnicity. Conclusions: No impact of ethnic background on the immune cell-mediated activity of the trifunctional antibody catumaxomab could be identified in terms of inducing potent pharmacological activity in vitro. Therefore, it is likely that patients of Asian ethnicity may also benefit from catumaxomab therapy as already demonstrated for Caucasian patients.

&lt;div&gt;Abstract&lt;p&gt;Fc engineering to enhance antibody effector functions harbors the potential to improve therapeutic effects. Understanding FcγR expression and distribution in the tumor microenvironment prior to and following treatment may help guide immune-engaging antibody design and patient stratification. In this study, we investigated FcR-expressing immune effector cells in HER2&lt;sup&gt;+&lt;/sup&gt; and triple-negative breast cancers (TNBC), including neoadjuvant chemotherapy–resistant disease. FcγRIIIa expression, FcγRIIIa&lt;sup&gt;+&lt;/sup&gt; NK cells, and classically activated (M1-like) macrophages correlated with improved anti-HER2 antibody efficacy. FcγRIIIa protein and FcγRIIIa&lt;sup&gt;+&lt;/sup&gt; NK cells and macrophages were present in primary TNBC and retained in treatment-resistant tumors. FcγRIIIa was spatially associated with folate receptor alpha–positive (FRα&lt;sup&gt;+&lt;/sup&gt;) tumor areas at baseline and in residual tumors following neoadjuvant chemotherapy. Wild-type and Fc-engineered antibodies recognizing two breast cancer–associated antigens, HER2 and the emerging TNBC target FRα, were designed and generated to have increased FcγRIIIa-expressing effector cell engagement. The combination of glycoengineering, including fucose removal from the N-linked Fc glycan, and Fc point mutations greatly increased antibody affinity for and retention on FcγRIIIa. The Fc-engineered antibodies enhanced immune effector activity against HER2&lt;sup&gt;+&lt;/sup&gt; breast cancer and TNBC, altering proinflammatory cytokine production by NK cells and tumor-conditioned macrophages and skewing macrophages toward proinflammatory states. Furthermore, the Fc-engineered antibodies restricted orthotopic HER2&lt;sup&gt;+&lt;/sup&gt; and FRα&lt;sup&gt;+&lt;/sup&gt; breast cancer xenograft growth at doses suboptimal for equivalent wild-type antibodies and recruited FcγRIIIa-expressing cells into tumors. Antibody design through combined glycoengineering and Fc point mutations to enhance FcγRIIIa engagement of tumor-infiltrating effector cells may be a promising strategy for developing therapies for patients with aggressive and treatment-resistant breast cancers.&lt;/p&gt;Significance:&lt;p&gt;Assessment of Fc receptors and immune cells in breast cancer enables development of tailored engineering strategies for tumor-targeting monoclonal antibodies with enhanced immune-stimulating and anticancer attributes by combining glycoengineering and Fc mutations.&lt;/p&gt;&lt;/div&gt;

Progress in cytokine engineering is driving therapeutic translation by overcoming these proteins' limitations as drugs. The IL-2 cytokine is a promising immune stimulant for cancer treatment but is limited by its concurrent activation of both pro-inflammatory immune effector cells and antiinflammatory regulatory T cells, toxicity at high doses, and short serum half-life. One approach to improve the selectivity, safety, and longevity of IL-2 is complexing with anti-IL-2 antibodies that bias the cytokine toward immune effector cell activation. Although this strategy shows potential in preclinical models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multiprotein drug and concerns regarding complex stability. Here, we introduced a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokine toward immune effector cells. We optimized IC construction and engineered the cytokine/antibody affinity to improve immune bias. We demonstrated that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared with natural IL-2, both alone and combined with immune checkpoint inhibitors. Moreover, therapeutic efficacy was observed without inducing toxicity. This work presents a roadmap for the design and translation of cytokine/antibody fusion proteins.

In Vitro and in Vivo Evidence of an Anti-Angiogenic Effect of Lenalidomide in Chronic Lymphocytic Leukemia

The clinical use of therapeutic antibodies has increased sharply because of their many advantages over conventional small molecule drugs, particularly with respect to their affinity, specificity, and serum stability. Tumor or infected cells are removed by the binding of antibody Fc regions to Fc gamma receptors (FcγRs), which stimulate the activation of immune effector cells. Aglycosylated full-length IgG antibodies expressed in bacteria have different Fc conformations compared to their glycosylated counterparts produced in mammalian cells. As a result, they are unable to bind FcγRs, resulting in little to no activation of immune effector cells. In this study, we created a combinatorial library randomized at the upper CH2 loops of an aglycosylated Fc variant (Fc5: E382V/M428) and used a high-throughput flow cytometry library screening method, combined with bacterial display of homodimeric Fc domains for enhanced FcγR binding affinity. The trastuzumab Fc variant containing the identified mutations (Q295R, L328W, A330V, P331A, I332Y, E382V, M428I) not only exhibited over 120 fold higher affinity of specific binding to FcγRI than wild type aglycosylated Fc, but also retained pH-dependent FcRn binding. These results show that an aglycosylated antibody expressed in bacteria can be evolved for novel FcγR affinity and specificity.

Since clinical experimental studies indicate that upper respiratory tract viral infections may exacerbate acute asthma symptoms in atopic/asthmatic individuals, we have investigated the expression and modulation of ICAM-1 on human nasal epithelial cells (HNEC) from normal and atopic subjects. ICAM-1 is the attachment molecule for the majority of serotypes of human rhinovirus (HRV), including HRV-14, and is also critical for the migration and activation of immune effector cells. Basal ICAM-1 expression was significantly higher in HNEC obtained by brushings from atopic compared with non-atopic subjects (P = 0.031), and was also significantly increased on atopic HNEC harvested in season compared with out of season (P < 0.05). Atopic HNEC showed further up-regulation in ICAM-1 expression when cultured with clinically relevant allergen (P = 0.032). ICAM-1 levels on normal HNEC were also increased by infection with HRV-14 (P < 0.05). Basal expression of ICAM-1 on atopic nasal polyp epithelial cells (EC) was significantly higher than on both normal and atopic nasal HNEC. This elevated nasal polyp ICAM-1 level was not increased further by allergen, although HRV infection resulted in a small significant increase. Recovered viral titres from HRV-infected nasal polyp EC were 1.5-fold higher than from infected normal nasal HNEC. The data are consistent with the hypothesis that allergen, by enhancing expression of the HRV attachment target on host cells, facilitates viral infection in atopic subjects; simultaneously HRV-induced increases in ICAM-1 levels would favour migration and activation of immune effector cells to the airway, resulting in enhanced atopic inflammation.

Oncoimmunologists are in a constant search for more effective immunotherapeutic treatments, helping to cure cancer. In this respect, immune cell participation is a key issue. A particularly interesting marker to identify antitumor immune cells is the neural cell adhesion molecule (NCAM), known as CD56. Namely, hematopoietic expression of CD56 seems to be confined to activated immune cells exhibiting some level of cytotoxic properties (Van Acker et al., Frontiers in Immunology 2017). Unfortunately, the current knowledge on the expression and functional role of CD56 is very fragmented. Therefore, we sought to elucidate the role of CD56 expression on various killer immune cells. First, we identified the high motility NCAM-120 isoform to be the main subset on immune cells. Next, through neutralization of surface CD56, we were able to demonstrate for the first time a direct involvement of CD56 in tumor cell lysis exerted by CD56-expressing killer cells such as natural killer cells, gamma delta (γδ) T-cells and interleukin (IL)-15-cultured dendritic cells (DCs). We also detected a putative crosstalk mechanism, suggesting CD56 as a co-stimulatory molecule in the interaction between IL-15 DCs and CD8 T-cells. Finally, by blocking the mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)–AKT pathway, with respectively trametinib and afuresertib, we confirmed our hypothesis that IL-15 stimulation directly leads to CD56 upregulation via the recruitment of shc, binding a phosphotyrosine residue on the IL-2/15Rβ chain. In conclusion, these results underscore the previously neglected importance of CD56 expression on immune cells, benefiting current and future immune therapeutic options. Citation Format: Heleen H. Van Acker, Maarten Versteven, Hans De Reu, Peter Ponsaerts, Zwi N Berneman, Viggo F. Van Tendeloo, Evelien L. Smits. CD56 participation in immune effector cell activation and tumor cell eradication: A role for interleukin-15 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B192.