A single bout of vigorous intensity exercise enhances the efficacy of rituximab against human chronic lymphocytic leukaemia B-cells ex vivo.

Chronic lymphocytic leukaemia (CLL) is characterised by the clonal proliferation and accumulation of mature B-cells and is often treated with rituximab, an anti-CD20 monoclonal antibody immunotherapy. Rituximab often fails to induce stringent disease eradication, due in part to failure of antibody-dependent cellular cytotoxicity (ADCC) which relies on natural killer (NK)-cells binding to rituximab-bound CD20 on B-cells. CLL cells are diffusely spread across lymphoid and other bodily tissues, and ADCC resistance in survival niches may be due to several factors including low NK-cell frequency and a suppressive stromal environment that promotes CLL cell survival. It is well established that exercise bouts induce a transient relocation of NK-cells and B-cells into peripheral blood, which could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab in blood. In this pilot study, n = 20 patients with treatment-naïve CLL completed a bout of cycling 15 % above anaerobic threshold for âˆ¼ 30-minutes, with blood samples collected pre-, immediately post-, and 1-hour post-exercise. Flow cytometry revealed that exercise evoked a 254 % increase in effector (CD3-CD56+CD16+) NK-cells in blood, and a 67 % increase in CD5+CD19+CD20+ CLL cells in blood (all p < 0.005). NK-cells were isolated from blood samples pre-, and immediately post-exercise and incubated with primary isolated CLL cells with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab-mediated cell lysis increased by 129 % following exercise (p < 0.001). Direct NK-cell lysis of CLL cells - independent of rituximab - was unchanged following exercise (p = 0.25). We conclude that exercise improved the efficacy of rituximab-mediated ADCC against autologous CLL cells ex vivo and propose that exercise should be explored as a means of enhancing clinical responses in patients receiving anti-CD20 immunotherapy.

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.

Agonist Antibodies for Cancer Immunotherapy: History, Hopes, and Challenges.

Immunotherapy is among the most promising new treatment modalities to arise over the last two decades; antibody drugs are delivering immunotherapy to millions of patients with many different types of cancer. Initial success with antibody therapeutics came in the form of direct targeting or cytotoxic antibodies, such as rituximab and trastuzumab, which bind directly to tumor cells to elicit their destruction. These were followed by immunomodulatory antibodies that elicit antitumor responses by either stimulating immune cells or relieving tumor-mediated suppression. By far the most successful approach in the clinic to date has been relieving immune suppression, with immune checkpoint blockade now a standard approach in the treatment of many cancer types. Despite equivalent and sometimes even more impressive effects in preclinical models, agonist antibodies designed to stimulate the immune system have lagged behind in their clinical translation. In this review, we document the main receptors that have been targeted by agonist antibodies, consider the various approaches that have been evaluated to date, detail what we have learned, and consider how their anticancer potential can be unlocked.

Human leukocyte immunoglobulin-like receptors in health and disease.

Human leukocyte immunoglobulin (Ig)-like receptors (LILR) are a family of 11 innate immunomodulatory receptors, primarily expressed on lymphoid and myeloid cells. LILRs are either activating (LILRA) or inhibitory (LILRB) depending on their associated signalling domains (D). With the exception of the soluble LILRA3, LILRAs mediate immune activation, while LILRB1-5 primarily inhibit immune responses and mediate tolerance. Abnormal expression and function of LILRs is associated with a range of pathologies, including immune insufficiency (infection and malignancy) and overt immune responses (autoimmunity and alloresponses), suggesting LILRs may be excellent candidates for targeted immunotherapies. This review will discuss the biology and clinical relevance of this extensive family of immune receptors and will summarise the recent developments in targeting LILRs in disease settings, such as cancer, with an update on the clinical trials investigating the therapeutic targeting of these receptors.

XPO1 inhibition sensitises CLL cells to NK cell mediated cytotoxicity and overcomes HLA-E expression.

The first-in-class inhibitor of exportin-1 (XPO1) selinexor is currently under clinical investigation in combination with the BTK inhibitor ibrutinib for patients with chronic lymphocytic leukaemia (CLL) or non-Hodgkin lymphoma. Selinexor induces apoptosis of tumour cells through nuclear retention of tumour suppressor proteins and has also recently been described to modulate natural killer (NK) cell and T cell cytotoxicity against lymphoma cells. Here, we demonstrate that XPO1 inhibition enhances NK cell effector function against primary CLL cells via downregulation of HLA-E and upregulation of TRAIL death receptors DR4 and DR5. Furthermore, selinexor potentiates NK cell activation against CLL cells in combination with several approved treatments; acalabrutinib, rituximab and obinutuzumab. We further demonstrate that lymph node associated signals (IL-4 + CD40L) inhibit NK cell activation against CLL cells via upregulation of HLA-E, and that inhibition of XPO1 can overcome this protective effect. These findings allow for the design of more efficacious combination strategies to harness NK cell effector functions against CLL.

Effect of posttranslational modifications and subclass on IgG activity: from immunity to immunotherapy.

Humoral immune responses are characterized by complex mixtures of polyclonal antibody species varying in their isotype, target epitope specificity and affinity. Posttranslational modifications occurring during antibody production in both the antibody variable and constant domain create further complexity and can modulate antigen specificity and antibody Fc-dependent effector functions, respectively. Finally, modifications of the antibody backbone after secretion may further impact antibody activity. An in-depth understanding of how these posttranslational modifications impact antibody function, especially in the context of individual antibody isotypes and subclasses, is only starting to emerge. Indeed, only a minute proportion of this natural variability in the humoral immune response is currently reflected in therapeutic antibody preparations. In this Review, we summarize recent insights into how IgG subclass and posttranslational modifications impact IgG activity and discuss how these insights may be used to optimize therapeutic antibody development.

Delivering co-stimulatory tumor necrosis factor receptor agonism for cancer immunotherapy: past, current and future perspectives.

The tumor necrosis factor superfamily (TNFSF) and their receptors (TNFRSF) are important regulators of the immune system, mediating proliferation, survival, differentiation, and function of immune cells. As a result, their targeting for immunotherapy is attractive, although to date, under-exploited. In this review we discuss the importance of co-stimulatory members of the TNFRSF in optimal immune response generation, the rationale behind targeting these receptors for immunotherapy, the success of targeting them in pre-clinical studies and the challenges in translating this success into the clinic. The efficacy and limitations of the currently available agents are discussed alongside the development of next generation immunostimulatory agents designed to overcome current issues, and capitalize on this receptor class to deliver potent, durable and safe drugs for patients.

Development of C1q Affinity Chromatography for the Study of C1q-IgG Interactions.

The classical complement system represents a central effector mechanism of Abs initiated by the binding of C1q to target bound IgG. Human C1q contains six heterotrimeric globular head groups that mediate IgG interaction, resulting in an avidity-driven binding event involving multiple IgG molecules binding a single C1q. Accordingly, surface bound IgG molecules are thought to assemble into noncovalent hexameric rings for optimal binding to the six-headed C1q. To study the C1q-Fc interaction of various Abs and screen for altered C1q binding mutants, we developed, to our knowledge, a novel HPLC-based method. Employing a single-chain form of C1q representing one C1q head group, our HPLC methodology was able to detect the interaction between the single-chain monomeric form of C1q and various ligands. We show that, despite a narrow window of specific binding owing to the low affinity of the monomeric C1q-IgG interaction, this approach clearly distinguished between IgG subclasses with established C1q binding properties. IgG3 displayed the strongest binding, followed by IgG1, with IgG2 and IgG4 showing the weakest binding. Fc mutants known to have increased C1q binding through oligomerization or enhanced C1q interaction showed greatly increased column retention, and IgG glycovariants displayed a consistent trend of increasing retention upon increasing galactosylation and sialylation. Furthermore, the column retention of IgG isotypes and glycovariants matches both the cell surface recruitment of C1q and complement-mediated cytotoxicity induced by each variant on an anti-CD20 Ab backbone. This methodology therefore provides a valuable tool for testing IgG Ab (glyco)variants for C1q binding, with clear relevance for therapeutic Ab development.

Reducing affinity as a strategy to boost immunomodulatory antibody agonism.

Antibody responses during infection and vaccination typically undergo affinity maturation to achieve high-affinity binding for efficient neutralization of pathogens1,2. Similarly, high affinity is routinely the goal for therapeutic antibody generation. However, in contrast to naturally occurring or direct-targeting therapeutic antibodies, immunomodulatory antibodies, which are designed to modulate receptor signalling, have not been widely examined for their affinity-function relationship. Here we examine three separate immunologically important receptors spanning two receptor superfamilies: CD40, 4-1BB and PD-1. We show that low rather than high affinity delivers greater activity through increased clustering. This approach delivered higher immune cell activation, in vivo T cell expansion and antitumour activity in the case of CD40. Moreover, an inert anti-4-1BB monoclonal antibody was transformed into an agonist. Low-affinity variants of the clinically important antagonistic anti-PD-1 monoclonal antibody nivolumab also mediated more potent signalling and affected T cell activation. These findings reveal a new paradigm for augmenting agonism across diverse receptor families and shed light on the mechanism of antibody-mediated receptor signalling. Such affinity engineering offers a rational, efficient and highly tuneable solution to deliver antibody-mediated receptor activity across a range of potencies suitable for translation to the treatment of human disease.

Hinge disulfides in human IgG2 CD40 antibodies modulate receptor signaling by regulation of conformation and flexibility.

Antibodies protect from infection, underpin successful vaccines and elicit therapeutic responses in otherwise untreatable cancers and autoimmune conditions. The human IgG2 isotype displays a unique capacity to undergo disulfide shuffling in the hinge region, leading to modulation of its ability to drive target receptor signaling (agonism) in a variety of important immune receptors, through hitherto unexplained molecular mechanisms. To address the underlying process and reveal how hinge disulfide orientation affects agonistic activity, we generated a series of cysteine to serine exchange variants in the hinge region of the clinically relevant monoclonal antibody ChiLob7/4, directed against the key immune receptor CD40. We report how agonistic activity varies with disulfide pattern and is afforded by the presence of a disulfide crossover between F(ab) arms in the agonistic forms, independently of epitope, as observed in the determined crystallographic structures. This structural "switch" affects directly on antibody conformation and flexibility. Small-angle x-ray scattering and ensemble modeling demonstrated that the least flexible variants adopt the fewest conformations and evoke the highest levels of receptor agonism. This covalent change may be amenable for broad implementation to modulate receptor signaling in an epitope-independent manner in future therapeutics.

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.

KIR2DS2 Expression Identifies NK Cells With Enhanced Anticancer Activity.

NK cells are promising cellular therapeutics against hematological and solid malignancies. Immunogenetic studies have identified that various activating killer cell Ig-like receptors (KIRs) are associated with cancer outcomes. Specifically, KIR2DS2 has been associated with reduced incidence of relapse following transplant in hematological malignancies and improved outcomes in solid tumors, but the mechanism remains obscure. Therefore, we investigated how KIR2DS2 expression impacts NK cell function. Using a novel flow cytometry panel, we show that human NK cells with high KIR2DS2 expression have enhanced spontaneous activation against malignant B cell lines, liver cancer cell lines, and primary chronic lymphocytic leukemia cells. Surface expression of CD16 was increased on KIR2DS2high NK cells, and, accordingly, KIR2DS2high NK cells had increased activation against lymphoma cells coated with the clinically relevant anti-CD20 Abs rituximab and obinutuzumab. Bulk RNA sequencing revealed that KIR2DS2high NK cells have upregulation of NK-mediated cytotoxicity, translation, and FCGR gene pathways. We developed a novel single-cell RNA-sequencing technique to identify KIR2DS2+ NK cells, and this confirmed that KIR2DS2 is associated with enhanced NK cell-mediated cytotoxicity. This study provides evidence that KIR2DS2 marks a population of NK cells primed for anticancer activity and indicates that KIR2DS2 is an attractive target for NK-based therapeutic strategies.

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.

Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc-engineering.

Agonistic CD27 monoclonal antibodies (mAb) have demonstrated impressive anti-tumour efficacy in multiple preclinical models but modest clinical responses. This might reflect current reagents delivering suboptimal CD27 agonism. Here, using a novel panel of CD27 mAb including a clinical candidate, we investigate the determinants of CD27 mAb agonism. Epitope mapping and in silico docking analysis show that mAb binding to membrane-distal and external-facing residues are stronger agonists. However, poor epitope-dependent agonism could partially be overcome by Fc-engineering, using mAb isotypes that promote receptor clustering, such as human immunoglobulin G1 (hIgG1, h1) with enhanced affinity to Fc gamma receptor (FcγR) IIb, or hIgG2 (h2). This study provides the critical knowledge required for the development of agonistic CD27 mAb that are potentially more clinically efficacious.

Fc-null anti-PD-1 monoclonal antibodies deliver optimal checkpoint blockade in diverse immune environments.

BACKGROUND: Despite extensive clinical use, the mechanisms that lead to therapeutic resistance to anti-programmed cell-death (PD)-1 monoclonal antibodies (mAbs) remain elusive. Here, we sought to determine how interactions between the Fc region of anti-PD-1 mAbs and Fcγ receptors (FcγRs) affect therapeutic activity and how these are impacted by the immune environment. METHODS: Mouse and human anti-PD-1 mAbs with different Fc binding profiles were generated and characterized in vitro. The ability of these mAbs to elicit T-cell responses in vivo was first assessed in a vaccination setting using the model antigen ovalbumin. The antitumor activity of anti-PD-1 mAbs was investigated in the context of immune 'hot' MC38 versus 'cold' neuroblastoma tumor models, and flow cytometry performed to assess immune infiltration. RESULTS: Engagement of activating FcγRs by anti-PD-1 mAbs led to depletion of activated CD8 T cells in vitro and in vivo, abrogating therapeutic activity. Importantly, the extent of this Fc-mediated modulation was determined by the surrounding immune environment. Low FcγR-engaging mouse anti-PD-1 isotypes, which are frequently used as surrogates for human mAbs, were unable to expand ovalbumin-reactive CD8 T cells, in contrast to Fc-null mAbs. These results were recapitulated in mice expressing human FcγRs, in which clinically relevant hIgG4 anti-PD-1 led to reduced endogenous expansion of CD8 T cells compared with its engineered Fc-null counterpart. In the context of an immunologically 'hot' tumor however, both low-engaging and Fc-null mAbs induced long-term antitumor immunity in MC38-bearing mice. Finally, a similar anti-PD-1 isotype hierarchy was demonstrated in the less responsive 'cold' 9464D neuroblastoma model, where the most effective mAbs were able to delay tumor growth but could not induce long-term protection. CONCLUSIONS: Our data collectively support a critical role for Fc:FcγR interactions in inhibiting immune responses to both mouse and human anti-PD-1 mAbs, and highlight the context-dependent effect that anti-PD-1 mAb isotypes can have on T-cell responses. We propose that engineering of Fc-null anti-PD-1 mAbs would prevent FcγR-mediated resistance in vivo and allow maximal T-cell stimulation independent of the immunological environment.

Disparity in peripheral and renal B-cell depletion with rituximab in systemic lupus erythematosus: an opportunity for obinutuzumab?

OBJECTIVES: To investigate key factors that may contribute to the variability of rituximab-mediated peripheral and renal B cell depletion (BCD) in SLE. METHODS: We analysed: (i) CD19+ B cell counts in patients with SLE before and 1, 2, 3 and 6 months after treatment with rituximab, comparing them with RA patients; (ii) the presence of B cells in renal biopsies after rituximab therapy; (iii) whether the duration of BCD correlated with patient demographics and B cell expression of CD20 and FcγRIIb; and (iv) the effect of B cell activation factor (BAFF) on the efficiency of rituximab and obinutuzumab at inducing BCD in whole blood assays, in vitro. RESULTS: In SLE (n = 71), the duration of BCD was shorter compared with RA (n = 27). B cells were detectable in renal biopsy samples (n = 6) after treatment with rituximab in all patients with poor response while peripheral blood B cells remained low or undetectable in the same patients. There were no significant relationships between peripheral BCD and patient age, disease duration, serum C3 levels or the level of expression of B cell surface proteins CD20 and FcγRIIb. Obinutuzumab was more efficient than rituximab at inducing BCD in whole blood assays, regardless of excess BAFF. CONCLUSIONS: BCD in SLE is less efficient than in RA. Renal B cell presence following rituximab treatment was associated with poor outcomes. No significant relationships between any measured B cell related, clinical or laboratory parameters and the efficiency of BCD by rituximab was found. Obinutuzumab was superior to rituximab at inducing BCD.

Paradoxes of cancer: Survival at the brink.

The fundamental understanding of how Cancer initiates, persists and then progresses is evolving. High-resolution technologies, including single-cell mutation and gene expression measurements, are now attainable, providing an ever-increasing insight into the molecular details. However, this higher resolution has shown that somatic mutation theory itself cannot explain the extraordinary resistance of cancer to extinction. There is a need for a more Systems-based framework of understanding cancer complexity, which in particular explains the regulation of gene expression during cell-fate decisions. Cancer displays a series of paradoxes. Here we attempt to approach them from the view-point of adaptive exploration of gene regulatory networks at the edge of order and chaos, where cell-fate is changed by oscillations between alternative regulators of cellular senescence and reprogramming operating through self-organisation. On this background, the role of polyploidy in accessing the phylogenetically pre-programmed "oncofetal attractor" state, related to unicellularity, and the de-selection of unsuitable variants at the brink of cell survival is highlighted. The concepts of the embryological and atavistic theory of cancer, cancer cell "life-cycle", and cancer aneuploidy paradox are dissected under this lense. Finally, we challenge researchers to consider that cancer "defects" are mostly the adaptation tools of survival programs that have arisen during evolution and are intrinsic of cancer. Recognition of these features should help in the development of more successful anti-cancer treatments.