CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

Cancer Immunosurveillance Caught in the Act.

The role of the immune system in the control of cancer formation and progression has been a matter of considerable debate over many years. In this issue of Immunity, Mlecnik et al. (2016) show the importance of immunosurveillance in controlling tumors in a series of microsatellite-instable human colon carcinomas.

Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy.

Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.

A human IgE bispecific antibody shows potent cytotoxic capacity mediated by monocytes.

The generation of bispecific antibodies (bsAbs) targeting two different antigens opens a new level of specificity and, compared to mAbs, improved clinical efficacy in cancer therapy. Currently, the different strategies for development of bsAbs primarily focus on IgG isotypes. Nevertheless, in comparison to IgG isotypes, IgE has been shown to offer superior tumor control in preclinical models. Therefore, in order to combine the promising potential of IgE molecules with increased target selectivity of bsAbs, we developed dual tumor-associated antigen-targeting bispecific human IgE antibodies. As proof of principle, we used two different pairing approaches - knobs-into-holes and leucine zipper-mediated pairing. Our data show that both strategies were highly efficient in driving bispecific IgE formation, with no undesired pairings observed. Bispecific IgE antibodies also showed a dose-dependent binding to their target antigens, and cell bridging experiments demonstrated simultaneous binding of two different antigens. As antibodies mediate a major part of their effector functions through interaction with Fc receptors (FcRs) expressed on immune cells, we confirmed FcεR binding by inducing in vitro mast cell degranulation and demonstrating in vitro and in vivo monocyte-mediated cytotoxicity against target antigen-expressing Chinese hamster ovary cells. Moreover, we demonstrated that the IgE bsAb construct was significantly more efficient in mediating antibody-dependent cell toxicity than its IgG1 counterpart. In conclusion, we describe the successful development of first bispecific IgE antibodies with superior antibody-dependent cell toxicity-mediated cell killing in comparison to IgG bispecific antibodies. These findings highlight the relevance of IgE-based bispecific antibodies for clinical application.

Identification and experimental validation of druggable epigenetic targets in hepatoblastoma.

BACKGROUND & AIMS: Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. METHODS: We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n = 72) and tumoral (n = 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. RESULTS: Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of ß-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. CONCLUSIONS: HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. IMPACT AND IMPLICATIONS: In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.

Differential Interleukin-8 thresholds for chemotaxis and netosis in human neutrophils.

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.

Anti-TGFß (Transforming Growth Factor ß) Therapy With Betaglycan-Derived P144 Peptide Gene Delivery Prevents the Formation of Aortic Aneurysm in a Mouse Model of Marfan Syndrome.

Objective: We investigated the effect of a potent TGFß (transforming growth factor ß) inhibitor peptide (P144) from the betaglycan/TGFß receptor III on aortic aneurysm development in a Marfan syndrome mouse model. Approach and Results: We used a chimeric gene encoding the P144 peptide linked to apolipoprotein A-I via a flexible linker expressed by a hepatotropic adeno-associated vector. Two experimental approaches were performed: (1) a preventive treatment where the vector was injected before the onset of the aortic aneurysm (aged 4 weeks) and followed-up for 4 and 20 weeks and (2) a palliative treatment where the vector was injected once the aneurysm was formed (8 weeks old) and followed-up for 16 weeks. We evaluated the aortic root diameter by echocardiography, the aortic wall architecture and TGFß signaling downstream effector expression of pSMAD2 and pERK1/2 by immunohistomorphometry, and Tgfß1 and Tgfß2 mRNA expression levels by real-time polymerase chain reaction. Marfan syndrome mice subjected to the preventive approach showed no aortic dilation in contrast to untreated Marfan syndrome mice, which at the same end point age already presented the aneurysm. In contrast, the palliative treatment with P144 did not halt aneurysm progression. In all cases, P144 improved elastic fiber morphology and normalized pERK1/2-mediated TGFß signaling. Unlike the palliative treatment, the preventive treatment reduced Tgfß1 and Tgfß2 mRNA levels. Conclusions: P144 prevents the onset of aortic aneurysm but not its progression. Results indicate the importance of reducing the excess of active TGFß signaling during the early stages of aortic disease progression.

Modulation of intratumoural myeloid cells, the hallmark of the anti-tumour efficacy induced by a triple combination: tumour-associated peptide, TLR-3 ligand and α-PD-1.

BACKGROUND: Anti-programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) monoclonal antibodies (mAbs) show remarkable clinical anti-tumour efficacy. However, rational combinations are needed to extend the clinical benefit to primary resistant tumours. The design of such combinations requires the identification of the kinetics of critical immune cell populations in the tumour microenvironment. METHODS: In this study, we compared the kinetics of immune cells in the tumour microenvironment upon treatment with immunotherapy combinations with different anti-tumour efficacies in the non-inflamed tumour model TC-1/A9. Tumour-bearing C57BL/6J mice were treated with all possible combinations of a human papillomavirus (HPV) E7 long peptide, polyinosinic-polycytidylic acid (PIC) and anti-PD-1 mAb. Tumour growth and kinetics of the relevant immune cell populations were assessed over time. The involvement of key immune cells was confirmed by depletion with mAbs and immunophenotyping with multiparametric flow cytometry. RESULTS: The maximum anti-tumour efficacy was achieved after intratumoural administration of HPV E7 long peptide and PIC combined with the systemic administration of anti-PD-1 mAb. The intratumoural immune cell kinetics of this combination was characterised by a biphasic immune response. An initial upsurge of proinflammatory myeloid cells led to a further rise in effector CD8+ T lymphocytes at day 8. Depletion of either myeloid cells or CD8+ T lymphocytes diminished the anti-tumour efficacy of the combination. CONCLUSIONS: The anti-tumour efficacy of a successful immunotherapy combination in a non-inflamed tumour model relies on an early inflammatory process that remodels the myeloid cell compartment.

Treatment of Experimental Autoimmune Encephalomyelitis by Sustained Delivery of Low-Dose IFN-α.

Multiple sclerosis (MS) is a chronic autoimmune disease with no curative treatment. The immune regulatory properties of type I IFNs have led to the approval of IFN-ß for the treatment of relapsing-remitting MS. However, there is still an unmet need to improve the tolerability and efficacy of this therapy. In this work, we evaluated the sustained delivery of IFN-α1, either alone or fused to apolipoprotein A-1 by means of an adeno-associated viral (AAV) system in the mouse model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. These in vivo experiments demonstrated the prophylactic and therapeutic efficacy of the AAV-IFN-α or AAV-IFN-α fused to apolipoprotein A-1 vectors in experimental autoimmune encephalomyelitis, even at low doses devoid of hematological or neurologic toxicity. The sustained delivery of such low-dose IFN-α resulted in immunomodulatory effects, consisting of proinflammatory monocyte and T regulatory cell expansion. Moreover, encephalitogenic T lymphocytes from IFN-α-treated mice re-exposed to the myelin oligodendrocyte glycoprotein peptide in vitro showed a reduced proliferative response and cytokine (IL-17A and IFN-γ) production, in addition to upregulation of immunosuppressive molecules, such as IL-10, IDO, or PD-1. In conclusion, the results of the present work support the potential of sustained delivery of low-dose IFN-α for the treatment of MS and likely other T cell-dependent chronic autoimmune disorders.

Dual activity of PD-L1 targeted Doxorubicin immunoliposomes promoted an enhanced efficacy of the antitumor immune response in melanoma murine model.

BACKGROUND: The immunomodulation of the antitumor response driven by immunocheckpoint inhibitors (ICIs) such as PD-L1 (Programmed Death Ligand-1) monoclonal antibody (α-PD-L1) have shown relevant clinical outcomes in a subset of patients. This fact has led to the search for rational combinations with other therapeutic agents such as Doxorubicin (Dox), which cytotoxicity involves an immune activation that may enhance ICI response. Therefore, this study aims to evaluate the combination of chemotherapy and ICI by developing Dox Immunoliposomes functionalized with monovalent-variable fragments (Fab') of α-PD-L1. RESULTS: Immunoliposomes were assayed in vitro and in vivo in a B16 OVA melanoma murine cell line over-expressing PD-L1. Here, immune system activation in tumor, spleen and lymph nodes, together with the antitumor efficacy were evaluated. Results showed that immunoliposomes bound specifically to PD-L1+ cells, yielding higher cell interaction and Dox internalization, and decreasing up to 30-fold the IC50, compared to conventional liposomes. This mechanism supported a higher in vivo response. Indeed, immunoliposomes promoted full tumor regression in 20% of mice and increased in 1 month the survival rate. This formulation was the only treatment able to induce significant (p < 0.01) increase of activated tumor specific cytotoxic T lymphocytes at the tumor site. CONCLUSION: PD-L1 targeted liposomes encapsulating Dox have proved to be a rational combination able to enhance the modulation of the immune system by blocking PD-L1 and selectively internalizing Dox, thus successfully providing a dual activity offered by both, chemo and immune therapeutic strategies.

Bioengineered PBGD variant improves the therapeutic index of gene therapy vectors for acute intermittent porphyria.

A first-in-human gene therapy trial using a recombinant adeno-associated viral (rAAV) vector for acute intermittent porphyria (AIP) reveals that higher doses would be required to reach therapeutic levels of the porphobilinogen deaminase (PBGD) transgene. We developed a hyperfunctional PBGD protein to improve the therapeutic index without increasing vector dose. A consensus protein sequence from 12 mammal species was compared to the human PBGD sequence, and eight amino acids were selected. I291M and N340S variants showed the highest increase in enzymatic activity when expressed in prokaryotic and eukaryotic systems. In silico analysis indicates that isoleucine 291 to methionine and asparagine 340 to serine variants did not affect the active site of the enzyme. In vitro analysis indicated a synergistic interaction between these two substitutions that improve kinetic stability. Finally, full protection against a phenobarbital-induced attack was achieved in AIP mice after the administration of 1 × 1011 gc/kg of rAAV2/8-PBGD-I291M/N340S vector; three times lower than the dose required to achieve full protection with the control rAAV2/8-hPBGD vector. In conclusion, we have developed and characterized a hyperfunctional PBGD protein. The inclusion of this variant sequence in a rAAV2/8 vector allows the effective dose to be lowered in AIP mice.

Innate immune mediators in cancer: between defense and resistance.

Chronic inflammation in the tumor microenvironment and evasion of the antitumor effector immune response are two of the emerging hallmarks required for oncogenesis and cancer progression. The innate immune system not only plays a critical role in perpetuating these tumor-promoting hallmarks but also in developing antitumor adaptive immune responses. Thus, understanding the dual role of the innate system in cancer immunology is required for the design of combined immunotherapy strategies able to tackle established tumors. Here, we review recent advances in the understanding of the role of cell populations and soluble components of the innate immune system in cancer, with a focus on complement, the adapter molecule Stimulator of Interferon Genes, natural killer cells, myeloid cells, and B cells.

Messenger RNA therapy for rare genetic metabolic diseases.

Decades of intense research in molecular biology and biochemistry are fructifying in the emergence of therapeutic messenger RNAs (mRNA) as a new class of drugs. Synthetic mRNAs can be sequence optimised to improve translatability into proteins, as well as chemically modified to reduce immunogenicity and increase chemical stability using naturally occurring uridine modifications. These structural improvements, together with the development of safe and efficient vehicles that preserve mRNA integrity in circulation and allow targeted intracellular delivery, have paved the way for mRNA-based therapeutics. Indeed, mRNAs formulated into biodegradable lipid nanoparticles are currently being tested in preclinical and clinical studies for multiple diseases including cancer immunotherapy and vaccination for infectious diseases. An emerging application of mRNAs is the supplementation of proteins that are not expressed or are not functional in a regulated and tissue-specific manner. This so-called 'protein replacement therapy' could represent a solution for genetic metabolic diseases currently lacking effective treatments. Here we summarise this new class of drugs and discuss the preclinical evidence supporting the potential of liver-mediated mRNA therapy for three rare genetic conditions: methylmalonic acidaemia, acute intermittent porphyria and ornithine transcarbamylase deficiency.

Cytokines in clinical cancer immunotherapy.

Cytokines are soluble proteins that mediate cell-to-cell communication. Based on the discovery of the potent anti-tumour activities of several pro-inflammatory cytokines in animal models, clinical research led to the approval of recombinant interferon-alpha and interleukin-2 for the treatment of several malignancies, even if efficacy was only modest. These early milestones in immunotherapy have been followed by the recent addition to clinical practice of antibodies that inhibit immune checkpoints, as well as chimeric antigen receptor T cells. A renewed interest in the anti-tumour properties of cytokines has led to an exponential increase in the number of clinical trials that explore the safety and efficacy of cytokine-based drugs, not only as single agents, but also in combination with other immunomodulatory drugs. These second-generation drugs under clinical development include known molecules with novel mechanisms of action, new targets, and fusion proteins that increase half-life and target cytokine activity to the tumour microenvironment or to the desired effector immune cells. In addition, the detrimental activity of immunosuppressive cytokines can be blocked by antagonistic antibodies, small molecules, cytokine traps or siRNAs. In this review, we provide an overview of the novel trends in the cytokine immunotherapy field that are yielding therapeutic agents for clinical trials.

A new immune-nanoplatform for promoting adaptive antitumor immune response.

Immunoliposomes (ILs), obtained with monoclonal antibodies (mAbs) decorating the liposome surface, are used for cancer treatment. These mAbs provide the recognition of molecules upregulated in cancer cells, like Programmed Death-Ligand 1 (PD-L1), an immune-checkpoint involved in tumor resistance, forming a complex that blocks this molecule and thereby, induces antitumor immune response. This mechanism introduces a new concept for ILs. ILs coupled to anti-PD-L1 or its Fab' fragment have been developed and in vitro/in vivo characterized. Factors such as coupling methods, PEG density and ligand size were optimized. In vitro data showed that Fab'-ILs displayed the highest PD-L1 cell interaction, correlating with a higher in vivo tumor accumulation and an increase of effector cytotoxic CD8+ T cells, providing tumor shrinkage and total regression in 20% of mice. Therefore, a novel immune-nanoplatform able to modulate the immune system has been developed, allowing the encapsulation of several agents for combinatorial therapies.

Exploiting scavenger receptors in cancer immunotherapy: Lessons from CD5 and SR-B1.

Scavenger receptors (SRs) are structurally heterogeneous cell surface receptors characterized by their capacity to remove extraneous or modified self-macromolecules from circulation, thus avoiding the accumulation of noxious agents in the extracellular space. This scavenging activity makes SRs important molecules for host defense and homeostasis. In turn, SRs keep the activation of the steady-state immune response in check, and participate as co-receptors in the priming of the effector immune responses when the macromolecules are associated with a threat that might compromise host homeostasis. Therefore, SRs built up sophisticated sensor mechanisms controlling the immune system, which may be exploited to develop novel drugs for cancer immunotherapy. In this review, we focus on the regulation of the anti-tumor immune response by two paradigmatic SRs: the lymphocyte receptor CD5 and the more broadly distributed scavenger receptor class B type 1 (SR-B1). Cancer immunity can be boosted by blockade of SRs working as immune checkpoint inhibitors (CD5) and/or by proper engagement of SRs working as innate danger receptor (SR-B1). Thus, these receptors illustrate both the complexity of targeting SRs in cancer immunotherapy and also the opportunities offered by such an approach.

Bile acids, FGF15/19 and liver regeneration: From mechanisms to clinical applications.

The liver has an extraordinary regenerative capacity rapidly triggered upon injury or resection. This response is intrinsically adjusted in its initiation and termination, a property termed the "hepatostat". Several molecules have been involved in liver regeneration, and among them bile acids may play a central role. Intrahepatic levels of bile acids rapidly increase after resection. Through the activation of farnesoid X receptor (FXR), bile acids regulate their hepatic metabolism and also promote hepatocellular proliferation. FXR is also expressed in enterocytes, where bile acids stimulate the expression of fibroblast growth factor 15/19 (FGF15/19), which is released to the portal blood. Through the activation of FGFR4 on hepatocytes FGF15/19 regulates bile acids synthesis and finely tunes liver regeneration as part of the "hepatostat". Here we review the experimental evidences supporting the relevance of the FXR-FGF15/19-FGFR4 axis in liver regeneration and discuss potential therapeutic applications of FGF15/19 in the prevention of liver failure. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Fibroblast growth factor 15/19 (FGF15/19) protects from diet-induced hepatic steatosis: development of an FGF19-based chimeric molecule to promote fatty liver regeneration.

OBJECTIVE: Fibroblast growth factor 15/19 (FGF15/19), an enterokine that regulates synthesis of hepatic bile acids (BA), has been proposed to influence fat metabolism. Without FGF15/19, mouse liver regeneration after partial hepatectomy (PH) is severely impaired. We studied the role of FGF15/19 in response to a high fat diet (HFD) and its regulation by saturated fatty acids. We developed a fusion molecule encompassing FGF19 and apolipoprotein A-I, termed Fibapo, and evaluated its pharmacological properties in fatty liver regeneration. DESIGN: Fgf15-/- mice were fed a HFD. Liver fat and the expression of fat metabolism and endoplasmic reticulum (ER) stress-related genes were measured. Influence of palmitic acid (PA) on FGF15/19 expression was determined in mice and in human liver cell lines. In vivo half-life and biological activity of Fibapo and FGF19 were compared. Hepatoprotective and proregenerative activities of Fibapo were evaluated in obese db/db mice undergoing PH. RESULTS: Hepatosteatosis and ER stress were exacerbated in HFD-fed Fgf15-/- mice. Hepatic expression of Pparγ2 was elevated in Fgf15-/- mice, being reversed by FGF19 treatment. PA induced FGF15/19 expression in mouse ileum and human liver cells, and FGF19 protected from PA-mediated ER stress and cytotoxicity. Fibapo reduced liver BA and lipid accumulation, inhibited ER stress and showed enhanced half-life. Fibapo provided increased db/db mice survival and improved regeneration upon PH. CONCLUSIONS: FGF15/19 is essential for hepatic metabolic adaptation to dietary fat being a physiological regulator of Pparγ2 expression. Perioperative administration of Fibapo improves fatty liver regeneration.

Antibody-dependent cell cytotoxicity: immunotherapy strategies enhancing effector NK cells.

Antibody-dependent cellular cytotoxicity (ADCC) is a set of mechanisms that target cells coated with IgG antibodies of the proper subclasses (IgG1 in the human) to be the prey of cell-to-cell cytolysis executed by immune cells expressing FcRIIIA (CD16A). These effectors include not only natural killer (NK) cells but also other CD16+ subsets such as monocyte/macrophages, NKT cells or γδ T cells. In cancer therapy, ADCC is exploited by antibodies that selectively recognize proteins on the surface of malignant cells. An approach to enhance antitumor activity is to act on effector cells so they are increased in their numbers or enhanced in their individual (on a cell per cell basis) ADCC performance. This enhancement can be therapeutically attained by cytokines (that is, interleukin (IL)-15, IL-21, IL-18, IL-2); immunostimulatory monoclonal antibodies (that is, anti-CD137, anti-CD96, anti-TIGIT, anti-KIR, anti-PD-1); TLR agonists or by adoptive infusions of ex vivo expanded NK cells which can be genetically engineered to become more efficient effectors. In conjunction with approaches optimizing IgG1 Fc affinity to CD16, acting on effector cells offers hope to achieve synergistic immunotherapy strategies.