TLR8激动剂GS-9688（selgantolimod）在慢性乙型肝炎中的治疗潜力

StephenW

Therapeutic potential of TLR8 agonist GS‐9688 (selgantolimod) in chronic hepatitis B: re‐modelling of antiviral and regulatory mediators
Dec 25 2020
Abstract
Background & Aims

GS‐9688 (selgantolimod) is a toll‐like receptor 8 (TLR8) agonist in clinical development for the treatment of chronic hepatitis B (CHB). Antiviral activity of GS‐9688 has previously been evaluated in vitro in hepatitis B virus (HBV)‐infected hepatocytes and in vivo in the woodchuck model of CHB. Here we evaluated the potential of GS‐9688 to boost responses contributing to viral control and to modulate regulatory mediators.
Approach & Results

We characterised the effect of GS‐9688 on immune cell subsets in vitro in PBMC of healthy controls and CHB patients. GS‐9688 activated dendritic cells and mononuclear phagocytes to produce IL‐12 and other immunomodulatory mediators, inducing a comparable cytokine profile in healthy controls and CHB patients. GS‐9688 increased the frequency of activated natural killer (NK) cells, mucosal‐associated invariant T‐cells (MAITs), CD4+ follicular helper T‐cells (TFH) and, in ~50% of patients, HBV‐specific CD8+T‐cells expressing interferon‐γ (IFNγ). Moreover, in vitro stimulation with GS‐9688 induced NK cell expression of IFNγ and TNFα and promoted hepatocyte lysis. We also assessed whether GS‐9688 inhibited immunosuppressive cell subsets that might enhance antiviral efficacy. Stimulation with GS‐9688 reduced the frequency of CD4+ regulatory T‐cells and monocytic myeloid‐derived suppressor cells (MDSC). Residual MDSC expressed higher levels of negative immune regulators, galectin‐9 and PD‐L1. Conversely, GS‐9688 induced an expansion of immunoregulatory TNF‐related apoptosis‐inducing ligand+ (TRAIL) regulatory NK cells and degranulation of arginase‐I+ polymorphonuclear‐MDSC (PMN‐MDSC).
Conclusions

GS‐9688 induces cytokines in human PBMC that are able to activate antiviral effector function by multiple immune mediators (HBV‐specific CD8+T‐cells, TFH, NK cells and MAITs). Whilst reducing the frequency of some immunoregulatory subsets, it enhances the immunosuppressive potential of others, highlighting potential biomarkers and immunotherapeutic targets to optimise the antiviral efficacy of GS‐9688.            

Introduction

Chronic hepatitis B (CHB) remains a global health concern with an estimated 260 million people infected worldwide. CHB causes more than 800,000 deaths a year due to hepatitis B virus (HBV)-related complications such as cirrhosis and hepatocellular carcinoma(1). In the absence of novel treatment strategies, it is projected that new cases of CHB will rise to three million per year by 2030(2). Current approved therapies include long term antiviral suppression with nucleos(t)ide analogues and pegylated interferon-alpha (IFN-α). These treatments reduce viremia and improve patient outcomes but are rarely curative(3). As a consequence, there is a pressing need for novel immunotherapeutic strategies to supplement existing direct-acting antivirals to achieve durable immune control.

Control of HBV is dependent on the co-ordinated action of both innate and adaptive immunity(4). A major obstacle to HBV clearance in CHB is a dysfunctional adaptive response, characterised by a profound state of immune exhaustion and HBV-specific T-cell depletion(5,6). The mechanism by which this dysfunction occurs is multi-faceted but likely driven by a combination of ongoing high-dose antigenic stimulation and the tolerogenic liver environment. Moreover, whilst the natural killer (NK) cell compartment can exert direct and indirect antiviral activity, they may also restrict effective antiviral immunity by deleting apoptosis-prone HBV-specific T-cells. Specifically, NK cells in the HBV-infected liver upregulate the death ligand TNF- related apoptosis-inducing ligand (TRAIL) enabling them to engage with and eliminate T-cells expressing the reciprocal receptor, TRAIL-receptor 2 (TRAIL-R2)(7,8). Another mechanism contributing to the suppression of HBV immunity is the expansion of arginase+ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) that deplete key nutrients required by T-cells for proliferation and function(9).

One therapeutic approach in clinical development for CHB is to engage innate immune receptors such as toll-like receptors (TLR), which can exert both direct and indirect effects on the antiviral T-cell and B-cell response. A previous therapeutic strategy attempted to harness TLR7 signalling using the agonist GS-9620 (vesatolimod). GS-9620 induced sustained antiviral responses in animal models(10,11), but failed to show therapeutic efficacy in CHB patients(12,13).

More recently, attention has turned to TLR8 activation due to its anticipated ability to stimulate host immunity through the induction of pro-inflammatory and immunomodulatory cytokines. Human TLR8 is predominantly expressed on the endosomal membrane of monocytes, macrophages, conventional dendritic cells (cDC) and CD4+ regulatory T-cells (TREG), and allows cells to respond to infection via detection of viral single-stranded RNA(14–16). GS-9688 (selgantolimod) is an oral selective small molecule agonist of TLR8 in development for CHB. In vitro studies have demonstrated that cytokines induced in human PBMC by GS-9688 reduce viral parameters in HBV-infected primary human hepatocytes(17). Furthermore, GS-9688 treatment was well tolerated and induced a sustained antiviral response in a subset of woodchuck hepatitis virus (WHV)-infected woodchucks(18). Although these preclinical studies provide some insight into the antiviral efficacy of GS-9688, they did not provide insight into the mode of action of GS-9688 on human immune cells.

Discussion

GS-9688 is a potent TLR8 agonist in development for the treatment of CHB. A recent study in the woodchuck model of CHB revealed that a short, finite period of dosing with GS-9688 is well tolerated and able to induce a sustained antiviral response(18). Here we evaluated the immunomodulatory effect of GS- 9688 on various immune cells in vitro using human PBMC to understand its therapeutic potential for CHB.

Initial experiments confirmed GS-9688 selectivity for TLR8, revealing the potent induction of immunomodulatory cytokines, such as IL-12, by cDC and MNP, which was preserved in PBMC from CHB patients. In interrogating the effects on correlates of immune protection, we showed that GS-9688 enhanced the frequency of HBV-specific CD8+T-cells in a subset of patients. In vitro treatment with GS- 9688 boosted the production of antiviral cytokines, but not cytotoxicity, of HBV-specific CD8+T-cells. The potential for GS-9688 to enhance cellular mediators with antiviral potential in CHB was further evidenced by increased activation, improved cytolytic and non-cytolytic effector function of NK cells and MAITs. In the HBV-infected liver, where NK cells are greatly enriched and where the virus-specific T-cell response is limited, it is possible that GS-9688-activated NK cells could perform a significant antiviral role through secretion of cytokines and enhanced killing of infected hepatocytes. In line with other TLR8 agonists, we confirmed the increase in antiviral activity by these effectors was driven by induction of IL-12/IL-18. IL-12 has the potential to reverse mitochondrial defects of exhausted HBV-specific CD8+T-cells allowing for more efficient bioenergetics and enhanced antiviral functionality(38–40). Importantly, the therapeutic benefit of IL-12 has been demonstrated in HBV transgenic mice, the woodchuck model of CHB and in early clinical trials in CHB patients(41–44).

Although promising, it is important to note that GS-9688 did not induce an effective HBV-specific CD8+T- cell response in PBMC from all patients, which is typical of many immunotherapeutic strategies in development for CHB using diverse patient cohorts(6,37,45). This is reminiscent of the antiviral response to GS-9688 in the woodchuck model of CHB, where only half the animals treated with 3mg/kg GS-9688 had a sustained reduction in viral parameters(18). Consistent with the woodchuck study, the response of patient HBV-specific CD8+T-cells in vitro was not associated with age, sex, extent of liver inflammation or viral load. However, the magnitude of the in vitro response was associated with a low baseline HBsAg titre (<2000IU/mL). To elucidate the potential differences in responders and non-responders, it is important to consider the contribution of potent immunoregulatory leukocytes implicated in suppressing T-cell immunity. In this study we show increased expression of the death ligand TRAIL on NK cells, particularly on the CD56bright subset driven by GS-9688, that may further eliminate the already depleted pool of HBV-specific T-cells within the infected liver(8). Conversely, these TRAIL+NK cells may act as anti-fibrotic mediators in CHB, killing activated hepatic stellate cells in a TRAIL-dependent manner or by producing hepatoprotective cytokines(46).

A notable finding of this study was GS-9688 modulation of immunosuppressive cells. GS-9688 reduced the frequency of TREG, potent immunosuppressors implicated in CHB(31). Intriguingly, the decline in TREG numbers was associated with a corresponding expansion and activation of cTFH. The observed GS-9866-driven increase in ICOS expression on cTFH is significant, as ICOS is critical for IL-21 production and the subsequent delivery of improved T-cell help(47,48). Although not studied here, we hypothesise that such an increase in cTFH and TFH-associated cytokines will improve immune control in CHB by modulating the frequency of memory B cells and production of affinity-matured class-switched antibodies. Whether this decrease in TREG and expansion and activation of cTFH is sufficient to overcome the observed B cell dysfunction characteristic of CHB patients will require further study(49,50).

Various other mechanisms have been identified that play a role in immune dysfunction in CHB, including the increased activity of MDSC. Consistent with the literature(35,36), we demonstrate the capacity of GS- 9688 to decrease the frequency of M-MDSC in vitro. However, this decrease was linked to an initial burst of arginase-I release from PMN-MDSC, which may transiently limit the availability of the conditionally essential amino acid L-arginine, acting as a crucial T-cell rheostat(9). Of further significance is the capacity of GS-9688 to induce expression of PD-L1 and galectin-9 on the remaining MDSC, which could limit HBV- specific CD8+T-cells expressing high levels of PD-1 and Tim-3(6,37). Intrahepatic PD-L1 and galectin-9 levels are increased in viral hepatitis, and our data suggest they may be further enhanced by TLR8-induced IFN. Collectively, these data raise the possibility that combination with checkpoint blockade may improve the antiviral response to GS-9688 treatment.

GS-9688 has good absorption and high first-pass hepatic clearance in order to limit systemic immune activation(17). After oral administration of GS-9688, intestinal absorption is expected to induce TLR8 activation in the gut and liver where the immune composition is markedly different(26,52–54). It is likely that the secretion of immune mediators from the gut into the portal vein will in turn stimulate cells in the liver. Given the fact that the liver harbours a population of transcriptionally distant liver-resident NK cells(26) and memory CD8+T-cells(53,54) future studies will need to focus on characterising the intrahepatic immune response to GS-9688.

Whilst the therapeutic efficacy of GS-9688 in combination with nucleos(t)ide treatment remains to be fully evaluated in CHB patients, it was shown to be safe and generally well-tolerated in a phase II placebo- controlled study in virally suppressed patients, with a subset achieving HBsAg and/or HBeAg loss(55). While phase II studies are ongoing (ClinicalTrials.gov ), our work provides important insights into the immunomodulatory effects of GS-9688 and has important implications for the rational design of combination studies with other immunomodulatory agents. Our data also raise the possibility that in vitro profiling of CHB patient PBMC responses to GS-9688 may have utility for predicting therapeutic efficacy in patients.

StephenW

TLR8激动剂GS-9688（selgantolimod）在慢性乙型肝炎中的治疗潜力：抗病毒和调节介质的重塑
2020年12月25日
抽象
背景与目标

GS-9688（selgantolimod）是临床上开发的收费型受体8（TLR8）激动剂，用于治疗慢性乙型肝炎（CHB）。先前已经在体外评估了乙型肝炎病毒（HBV）感染的肝细胞中和在CHB的土拨鼠模型中对GS‐9688的抗病毒活性进行了评估。在这里，我们评估了GS-9688增强应答有助于病毒控制和调节调节介质的潜力。
方法与结果

我们表征了健康对照组和CHB患者的PBMC中GS‐9688对免疫细胞亚群的体外作用。 GS-9688激活树突状细胞和单核吞噬细胞，产生IL-12和其他免疫调节介质，从而在健康对照和CHB患者中诱导出类似的细胞因子谱。 GS‐9688增加了活化的自然杀伤（NK）细胞，粘膜相关不变性T细胞（MAIT），CD4 +滤泡辅助性T细胞（TFH）的频率，在约50％的患者中，HBV特异性CD8 + T的频率增加表达干扰素γ（IFNγ）的细胞。此外，在体外用GS-9688刺激诱导NK细胞表达IFNγ和TNFα，并促进肝细胞裂解。我们还评估了GS-9688是否抑制了可能增强抗病毒功效的免疫抑制细胞亚群。 GS‐9688刺激降低了CD4 +调节性T细胞和单核细胞来源的抑制细胞（MDSC）的频率。残留的MDSC表达更高水平的阴性免疫调节剂，半乳凝素9和PD L1。相反，GS-9688诱导了免疫调节性TNF相关凋亡诱导配体+（TRAIL）调节性NK细胞的扩增，以及精氨酸酶I +多形核MDSC（PMN-MDSC）的脱粒。
结论

GS-9688诱导人PBMC中的细胞因子，该细胞因子能够通过多种免疫介质（HBV特异性CD8 + T细胞，TFH，NK细胞和MAITs）激活抗病毒效应子功能。在降低某些免疫调节子集的频率的同时，它增强了其他免疫调节子集的免疫抑制潜能，突出了潜在的生物标志物和免疫治疗靶标，以优化GS-9688的抗病毒功效。