Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function.

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.

Soluble CTLA-4 attenuates T cell activation and modulates anti-tumor immunity.

CTLA-4 is a crucial immune checkpoint receptor involved in the maintenance of immune homeostasis, tolerance, and tumor control. Antibodies targeting CTLA-4 have been promising treatments for numerous cancers, but the mechanistic basis of their anti-tumoral immune-boosting effects is poorly understood. Although the ctla4 gene also encodes an alternatively spliced soluble variant (sCTLA-4), preclinical/clinical evaluation of anti-CTLA-4-based immunotherapies have not considered the contribution of this isoform. Here, we explore the functional properties of sCTLA-4 and evaluate the efficacy of isoform-specific anti-sCTLA-4 antibody targeting in a murine cancer model. We show that expression of sCTLA-4 by tumor cells suppresses CD8+ T cells in vitro and accelerates growth and experimental metastasis of murine tumors in vivo. These effects were accompanied by modification of the immune infiltrate, notably restraining CD8+ T cells in a non-cytotoxic state. sCTLA-4 blockade with isoform-specific antibody reversed this restraint, enhancing intratumoral CD8+ T cell activation and cytolytic potential, correlating with therapeutic efficacy and tumor control. This previously unappreciated role of sCTLA-4 suggests that the biology and function of multi-gene products of immune checkpoint receptors need to be fully elucidated for improved mechanistic understanding of cancer immunotherapies.

Hiding in Plain Sight: Soluble Immunomodulatory Receptors.

Alternatively spliced natural soluble isoforms of immunomodulatory receptors [cytotoxic T lymphocyte antigen-4 (CTLA-4), 4-1BB, and programmed death-1 (PD-1)/PD-L1] have been overlooked in favor of their cell-surface-bound counterparts that have generated blockbuster antibodies for the treatment of cancer. We propose that the soluble variants of these receptors contribute to immune regulation and offer potential as targets for immunotherapy.

Improved Multiplex Immunohistochemistry for Immune Microenvironment Evaluation of Mouse Formalin-Fixed, Paraffin-Embedded Tissues.

Immune profiling of tissue through multiplex immunohistochemistry is important for the investigation of immune cell dynamics, and it can contribute to disease prognosis and evaluation of treatment response in cancer patients. However, protocols for mouse formalin-fixed, paraffin-embedded tissue have been less successful. Given that formalin fixation and paraffin embedding remains the most common preparation method for processing mouse tissue, this has limited the options to study the immune system and the impact of novel therapeutics in preclinical models. In an attempt to address this, we developed an improved immunohistochemistry protocol with a more effective Ag-retrieval buffer. We also validated 22 Abs specific for mouse immune cell markers to distinguish B cells, T cells, NK cells, macrophages, dendritic cells, and neutrophils. In addition, we designed and tested novel strategies to identify immune cells for which unique Abs are currently not available. Last, in the 4T1 model of breast cancer, we demonstrate the utility of our protocol and Ab panels in the quantitation and spatial distribution of immune cells.

Shaving Is an Epiphenomenon of Type I and II Anti-CD20-Mediated Phagocytosis, whereas Antigenic Modulation Limits Type I Monoclonal Antibody Efficacy.

Rituximab is an anti-CD20 mAb used in the treatment of B cell malignancies. Loss of surface CD20 Ag from the surface of target cells is thought to be one mechanism governing resistance to rituximab, but how this occurs is not completely understood. Two explanations for this have been proposed: antigenic modulation whereby mAb:CD20 complexes are internalized in a B cell intrinsic process and shaving, in which mAb:CD20 complexes undergo trogocytic removal by effector cells, such as macrophages. However, there is conflicting evidence as to which predominates in clinical scenarios and hence the best strategies to overcome resistance. In this study, we investigated the relative importance of modulation and shaving in the downregulation of surface mAb:CD20. We used both murine and human systems and treated ex vivo macrophages with varying concentrations of non-FcγR-interacting beads to achieve differential macrophage saturation states, hence controllably suppressing further phagocytosis of target cells. We then monitored the level and localization of mAb:CD20 using a quenching assay. Suppression of phagocytosis with bead treatment decreased shaving and increased modulation, suggesting that the two compete for surface rituximab:CD20. Under all conditions tested, modulation predominated in rituximab loss, whereas shaving represented an epiphenomenon to phagocytosis. We also demonstrate that the nonmodulating, glycoengineered, type II mAb obinutuzumab caused a modest but significant increase in shaving compared with type II BHH2 human IgG1 wild-type mAb. Therefore, shaving may represent an important mechanism of resistance when modulation is curtailed, and glycoengineering mAb to increase affinity for FcγR may enhance resistance because of shaving.

FcγR requirements leading to successful immunotherapy.

Monoclonal antibody (mAb) immunotherapy is currently experiencing an unprecedented amount of success, delivering blockbuster sales for the pharmaceutical industry. Having experienced several false dawns and overcoming technical issues which limited progress, we are now entering a golden period where mAbs are becoming a mainstay of treatment regimes for diseases ranging from cancer to autoimmunity. In this review, we discuss how these mAbs are most likely working and focus in particular on the key receptors that they interact with to precipitate their therapeutic effects. Although their targets may vary, their engagement with Fcγ receptors (FcγRs) on numerous immune effector cells is almost universal, and here we review their roles in delivering successful immunotherapy.

Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo.

Development and Characterization of Monoclonal Antibodies Specific for Mouse and Human Fcγ Receptors.

FcγRs are key regulators of the immune response, capable of binding to the Fc portion of IgG Abs and manipulating the behavior of numerous cell types. Through a variety of receptors, isoforms, and cellular expression patterns, they are able to fine-tune and direct appropriate responses. Furthermore, they are key determinants of mAb immunotherapy, with mAb isotype and FcγR interaction governing therapeutic efficacy. Critical to understanding the biology of this complex family of receptors are reagents that are robust and highly specific for each receptor. In this study, we describe the development and characterization of mAb panels specific for both mouse and human FcγR for use in flow cytometry, immunofluorescence, and immunocytochemistry. We highlight key differences in expression between the two species and also patterns of expression that will likely impact on immunotherapeutic efficacy and translation of therapeutic agents from mouse to clinic.

Rehabilitation or the death penalty: autoimmune B cells in the dock.

CD20-based monoclonal antibodies have become established as treatments for lymphoma, rheumatoid arthritis, systemic lupus erythematosus, vasculitis and dermatomyositis, with the principle therapeutic mechanism relating to B-cell depletion through effector cell engagement. An article by Brühl et al. in this issue of the European Journal of Immunology [Eur. J. Immunol. 2015. 45: 705-715] reveals a fundamentally distinct mechanism of silencing autoimmune B-cell responses. Rather than B-cell depletion, the authors use anti-CD79b antibodies to induce B-cell tolerance and suppress humoral immune responses against collagen to prevent the development of arthritis in mice. Here we highlight the differences in the mechanisms used by anti-CD20 and anti-CD79b Ab therapy and discuss why depletion of B cells may not be required to treat autoimmune arthritis and other B-cell-associated pathologies.

The soluble isoform of CTLA-4 as a regulator of T-cell responses.

CTLA-4 is a crucial immune regulator that mediates both negative costimulation signals to T cells, and regulatory T (Treg)-cell extrinsic control of effector responses. Here we present evidence supporting a novel mechanism for this extrinsic suppression, executed by the alternatively spliced soluble CTLA-4 isoform (sCTLA-4). Analyses of human T cells in vitro show that sCTLA-4 secretion can be increased during responses, and has potent inhibitory properties, since isoform-specific blockade of its activity significantly increased Ag-driven proliferation and cytokine (IFN-γ, IL-17) secretion. Treg cells were demonstrated to be a prominent source of sCTLA-4, which contributed to suppression in vitro when their numbers were limiting. The soluble isoform was also produced by, and inhibited, murine T cells responding to Ag in vitro, and blockade of its activity in vivo protected against metastatic spread of melanoma in mice. We conclude that sCTLA-4 is an important immune regulator, responsible for at least some of the inhibitory effects previously ascribed to the membrane-bound isoform. These results suggest that the immune system exploits the different CTLA-4 isoforms for either intrinsic or extrinsic regulation of T-cell activity.

Differential Regulation of the STING Pathway in Human Papillomavirus-Positive and -Negative Head and Neck Cancers.

Squamous cell carcinomas, which arise from the cells that line the mucosal surfaces of the head and neck, represent the most common type of head and neck cancers (HNSCC). Human papillomavirus (HPV) infection has been strongly associated with the development of oropharyngeal cancers, which are cancers that occur in the back of the throat, including the tonsils and base of the tongue. HNSCCs with and without HPV infection have distinct pathology, with HPV-positive patients having higher levels of immune infiltration, activation in the tumor microenvironment and better response to radiation and chemotherapy. It is, however, unclear whether HPV infection in HNSCCs has the potential to activate innate-immune sensing pathways and if these cancers possess intrinsic immunogenicity associated with HPV infection. Here we investigate the innate immune stimulator of interferon genes (STING) pathway and immune responses to STING activation in HNSCCs and uncover fundamental differences in the regulation of this pathway in cell lines versus primary human clinical specimens. We show that while STING is differentially expressed in HPV-positive and -negative HNSCC cell lines, they exhibit a gross functional defect in signaling through this pathway. However, STING activation in immune cell populations generated immune signatures predicted to elicit useful tumoricidal mechanisms. In contrast, IHC analysis of human tissue microarrays revealed enhanced STING expression in HPV-related tumors and high intratumoral expression of STING correlated with increased survival. SIGNIFICANCE: STING is an important innate immune sensor of cytosolic DNA, inducing essential antiviral and antitumoral responses. This research shows that STING expression is enhanced in HPV-positive HNSCC patient tissue, with high intratumoral STING expression correlating with increased survival. In addition, STING activation in immune cell populations augmented antitumoral effects against HNSCCs, suggesting patients may benefit from the use of STING agonists in combination with traditional therapies.

Neuroblastoma: an ongoing cold front for cancer immunotherapy.

Neuroblastoma is the most frequent extracranial childhood tumour but effective treatment with current immunotherapies is challenging due to its immunosuppressive microenvironment. Efforts to date have focused on using immunotherapy to increase tumour immunogenicity and enhance anticancer immune responses, including anti-GD2 antibodies; immune checkpoint inhibitors; drugs which enhance macrophage and natural killer T (NKT) cell function; modulation of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway; and engineering neuroblastoma-targeting chimeric-antigen receptor-T cells. Some of these strategies have strong preclinical foundation and are being tested clinically, although none have demonstrated notable success in treating paediatric neuroblastoma to date. Recently, approaches to overcome heterogeneity of neuroblastoma tumours and treatment resistance are being explored. These include rational combination strategies with the aim of achieving synergy, such as dual targeting of GD2 and tumour-associated macrophages or natural killer cells; GD2 and the B7-H3 immune checkpoint; GD2 and enhancer of zeste-2 methyltransferase inhibitors. Such combination strategies provide opportunities to overcome primary resistance to and maximize the benefits of immunotherapy in neuroblastoma.

Worked to the bone: antibody-based conditioning as the future of transplant biology.

Conditioning of the bone marrow prior to haematopoietic stem cell transplant is essential in eradicating the primary cause of disease, facilitating donor cell engraftment and avoiding transplant rejection via immunosuppression. Standard conditioning regimens, typically comprising chemotherapy and/or radiotherapy, have proven successful in bone marrow clearance but are also associated with severe toxicities and high incidence of treatment-related mortality. Antibody-based conditioning is a developing field which, thus far, has largely shown an improved toxicity profile in experimental models and improved transplant outcomes, compared to traditional conditioning. Most antibody-based conditioning therapies involve monoclonal/naked antibodies, such as alemtuzumab for graft-versus-host disease prophylaxis and rituximab for Epstein-Barr virus prophylaxis, which are both in Phase II trials for inclusion in conditioning regimens. Nevertheless, alternative immune-based therapies, including antibody-drug conjugates, radio-labelled antibodies and CAR-T cells, are showing promise in a conditioning setting. Here, we analyse the current status of antibody-based drugs in pre-transplant conditioning regimens and assess their potential in the future of transplant biology.

FcγRIIB controls antibody-mediated target cell depletion by ITIM-independent mechanisms.

Many therapeutic antibodies deplete target cells and elicit immunotherapy by engaging activating Fc gamma receptors (FcγRs) on host effector cells. These antibodies are negatively regulated by the inhibitory FcγRIIB (CD32B). Dogma suggests inhibition is mediated through the FcγRIIB immunoreceptor tyrosine-based inhibition motif (ITIM), negatively regulating immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling from activating FcγR. To assess this, we generated experimental models expressing human (h)FcγRIIB on targets or effectors, lacking or retaining ITIM signaling capacity. We demonstrate that signaling through the hFcγRIIB ITIM is dispensable for impairing monoclonal antibody (mAb)-mediated depletion of normal and malignant murine target cells through three therapeutically relevant surface receptors (CD20, CD25, and OX40) affecting immunotherapy. We demonstrate that hFcγRIIB competition with activating FcγRs for antibody Fc, rather than ITIM signaling, is sufficient to impair activating FcγR engagement, inhibiting effector function and immunotherapy.

HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy.

BACKGROUND: Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. METHODS: We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. RESULTS: We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. CONCLUSION: Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Investigating the Role of Indoleamine 2,3-Dioxygenase in Acute Myeloid Leukemia: A Systematic Review.

Background: The immunomodulatory enzyme, indoleamine 2,3-dioxygenase (IDO) facilitates tryptophan catabolism at the rate-limiting step of the kynurenine (Kyn) pathway. IDO expression and elevations in Kyn metabolites are associated with immunosuppressive tumor microenvironment including T cell proliferative arrest and generation of regulatory T cells (Tregs) which can favor tumor progression. However, the extent of the role of IDO in acute myeloid leukemia (AML) is currently ill-defined. This study reviews the role of IDO-driven Treg function in AML and evaluates the current body of evidence implicating IDO in AML pathogenesis. Method: Studies related to IDO in AML were identified through a systematic review of PubMed and Scopus. Data extracted described sample analysis, IDO expression, IDO in prognosis, techniques used in Treg phenotypic studies, and the effect of IDO inhibitors. Results: Twenty studies were included in the systematic review. Expression of IDO was identified in a range of cells in AML, both inducible and constitutive. Seven studies indicated an association between elevated expression and poor clinical prognosis. Six studies suggested a positive correlation between IDO expression and Treg induction, with FoxP3 being the prominent Treg phenotypic marker. Of eight studies investigating IDO inhibition, some reported reductions in Treg frequency and enhanced effector T cell proliferation. Conclusion: This review highlights that IDO expression in AML is associated with poor prognosis and measurement of IDO and its Kyn metabolites may offer utility as prospective prognostic markers. Pharmacological inhibition of IDO using novel drugs may hold promise for the treatment of AML.

Old Dog, New Trick: Type I IFN-Based Treatment for Acute Myeloid Leukemia.

Despite strong biological rationale for the use of type-I IFNs for the treatment of acute myeloid leukemia (AML), their usage is limited to few hematologic malignancies. Here, we propose that innate immune sensing machinery, particularly the stimulator of IFN genes pathway, may be exploited to deliver antileukemic effects in AML.

Recent Progress in Interferon Therapy for Myeloid Malignancies.

Myeloid malignancies are a heterogeneous group of clonal haematopoietic disorders, caused by abnormalities in haematopoietic stem cells (HSCs) and myeloid progenitor cells that originate in the bone marrow niche. Each of these disorders are unique and present their own challenges with regards to treatment. Acute myeloid leukaemia (AML) is considered the most aggressive myeloid malignancy, only potentially curable with intensive cytotoxic chemotherapy with or without allogeneic haematopoietic stem cell transplantation. In comparison, patients diagnosed with chronic myeloid leukaemia (CML) and treated with tyrosine kinase inhibitors (TKIs) have a high rate of long-term survival. However, drug resistance and relapse are major issues in both these diseases. A growing body of evidence suggests that Interferons (IFNs) may be a useful therapy for myeloid malignancies, particularly in circumstances where patients are resistant to existing front-line therapies and have risk of relapse following haematopoietic stem cell transplant. IFNs are a major class of cytokines which are known to play an integral role in the non-specific immune response. IFN therapy has potential as a combination therapy in AML patients to reduce the impact of minimal residual disease on relapse. Alongside this, IFNs can potentially sensitize leukaemic cells to TKIs in resistant CML patients. There is evidence also that IFNs have a therapeutic role in myeloproliferative neoplasms (MPNs) such as polycythaemia vera (PV) and primary myelofibrosis (PMF), where they can restore polyclonality in patients. Novel formulations have improved the clinical effectiveness of IFNs. Low dose pegylated IFN formulations improve pharmacokinetics and improve patient tolerance to therapies, thereby minimizing the risk of haematological toxicities. Herein, we will discuss recent developments and the current understanding of the molecular and clinical implications of Type I IFNs for the treatment of myeloid malignancies.

STINGing Viral Tumors: What We Know from Head and Neck Cancers.

It has now become increasingly clear that viruses, which may not be directly oncogenic, can affect the biology of tumors as well as immune behavior against tumors. This has led to a fundamental question: Should tumors associated with viral infection be considered distinct from those without? Typically, viruses activate the host innate immune responses by stimulating pathogen recognition receptors and DNA-sensing pathways, including the stimulator of interferon genes (STING) pathway. However, regulation of the STING pathway in a virus-associated tumor microenvironment is poorly understood. Human papillomavirus (HPV) infection within a subset of head and neck squamous cell carcinomas (HNSCC) promotes a unique etiology and clinical outcome. For reasons currently not well understood, patients with HPV+ tumors have a better outcome in terms of both overall survival and reduced risk of recurrence compared with HPV- HNSCC. This observation may reflect a greater intrinsic immunogenicity associated with HPV infection, pertaining to innate immune system pathways activated following recognition of viral nucleotides. Here we discuss how HNSCC provides a unique model to study the STING pathway in the context of viral-induced tumor type as well as recent advances in our understanding of this pathway in HSNCC.

Multi-platform profiling characterizes molecular subgroups and resistance networks in chronic lymphocytic leukemia.

Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches.