Engineered IFN-α and anti-PDL1 containing compounds to target the liver and restore antiviral protection for HBV cure

Chronic HBV infection, with hepatic inflammation and viral genome persistence in the hepatocytes either in the form of cccDNA or partially integrated into the host nucleus, is one of the major causes of liver cirrhosis and hepatocellular carcinoma. Available therapies for chronic hepatitis B (CHB) include longterm administration of nucleos(t)ide analogues or a finite treatment with polyethyleneglycol (PEG)–interferon alpha (IFN-α) that rarely allow reaching a functional cure, defined as loss of Hepatitis B surface antigen (HBsAg) and undetectable HBV DNA in the serum. Thus, a major effort is currently devoted to studies aimed at finding new effective therapies. Different factors contribute to viral persistence. HBV can elude the host immune response, being poorly detected by the innate system and inducing variable degrees of dysfunction of the adaptive immune response up to Tcell and Bcell exhaustion in chronic infection. Moreover, the liver represents a peculiar tolerogenic environment that plays a central role in the induction of immune unresponsiveness due to its unique anatomy and composition of resident cell population, as well as to the presence of different regulatory mechanisms, including high expression of coinhibitory receptors by immune cells. Therefore, strategies aimed at activating innate immunity components or at reconstituting an efficient antiviral Tcell and Bcell response represent a rational approach to allow virus control and to complement the effect of available antiviral therapies. For this reason, immunotherapy represents a rationale option among many different therapeutic approaches currently under investigation. The rationale for the use of IFN-α is based not only on its direct antiviral effect but also on its immunomodulatory properties. IFN-α signalling leads to the induction of more than 300 interferonstimulated genes (ISG), most of which encode patternrecognition receptors that detect viral molecules, while some of them encode proteins that act as antiviral effectors in both infected and noninfected cells, with the aim to limit viral replication and spread. Besides the induction of ISG expression, a direct antiviral effect is sustained through different additional mechanisms including the induction of cytidine deamination and HBV cccDNA degradation, inhibition of viral capsid assembly and of cccDNA transcription, accelerated pregenomic RNA and core particle degradation. Among the immunological properties of IFN-α, its boosting effect on the innate response, including improvement of natural killer (NK) cell antiviral function and dendritic cell (DC) crosspriming, is pivotal for a successful response to IFN-α treatment. Conversely, the effect of IFN-α is modest or even inhibitory on HBVspecific CD8 Tcell responses. In order to improve the pharmacokinetics and prolong its biological activity, IFN-α is currently used conjugated to PEG. However, PEG–IFN-α administration alone allows achievement of stable HBsAg loss in about 5%–10% of treated patients with CHB, and it is not well tolerated by all patients because of a number of frequent adverse events. With the aim to find a strategy to reduce IFN-α side effects deriving from systemic administration, IFN-α was engineered to specifically target the liver by its fusion to Tcell receptorlike (TCRL) antibodies able to selectively recognise viral peptides/ human leucocyte antigen (HLA) complexes on HBV infected cells. These modifications allowed to focus a full IFN-α activity specifically on cells expressing the appropriate HLA–peptide complex, increasing the locally achievable drug concentration, with possible reduction of the administered dose, and consequently of systemic toxicity. However, several limitations hampered the possibility to test the TCRL–IFN-α in animals, such as the HLA class I specificity of the molecule and the lack of crossreactivity between human IFN-α with mouse receptors. The possibility of IFN type I local delivery has been widely investigated in the setting of antitumour therapies, where, among different formulations, conjugation with an anti-PDL1 antibody has been tested in order to target IFN-α to PDL1expressing cells in the tumour