Preclinical risk assessment strategy to mitigate the T-cell dependent immunogenicity of protein biotherapeutics: State of the art, challenges and future perspectives.

Protein therapeutics hold a prominent role and have brought significant diversity in efficacious medicinal products. Not just monoclonal antibodies and different antibody formats (pegylated antigen-binding fragments, bispecifics, antibody-drug conjugates, single chain variable fragments, nanobodies, dia-, tria- and tetrabodies), but also purified blood products, growth factors, recombinant cytokines, enzyme replacement factors, fusion proteins are all good instances of therapeutic proteins that have been developed in the past decades and approved for their value in oncology, immune-oncology, and autoimmune diseases discovery programs. Although there was an ingrained belief that fully humanized proteins were expected to have limited immunogenicity, adverse effects associated with immune responses to biological therapies raised some concern in biotech companies. Consequently, drug developers are designing strategies to assess potential immune responses to protein therapeutics during both the preclinical and clinical phases of development. Despite the many factors that can contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) immunogenicity seems to play a crucial role in the development of anti-drug antibodies (ADAs) to biologics. A broad range of methodologies to predict and rationally assess Td immune responses to protein drugs has been developed. This review aims to briefly summarize the preclinical immunogenicity risk assessment strategy to mitigate the risk of potential immunogenic candidates coming towards clinical phases, discussing the advantages and limitations of these technologies, and suggesting a rational approach for assessing and mitigating Td immunogenicity.

Survival and migration of human dendritic cells are regulated by an IFN-alpha-inducible Axl/Gas6 pathway.

Axl, a prototypic member of the transmembrane tyrosine kinase receptor family, is known to regulate innate immunity. In this study, we show that Axl expression is induced by IFN-alpha during human dendritic cell (DC) differentiation from monocytes (IFN/DC) and that constitutively Axl-negative, IL-4-differentiated DC (IL-4/DC) can be induced to up-regulate Axl by IFN-alpha. This effect is inhibited by TLR-dependent maturation stimuli such as LPS, poly(I:C), TLR7/8 ligand, and CD40L. LPS-induced Axl down-regulation on the surface of human IFN-alpha-treated DC correlates with an increased proteolytic cleavage of Axl and with elevated levels of its soluble form. GM6001 and TAPI-1, general inhibitors of MMP and ADAM family proteases, restored Axl expression on the DC surface and diminished Axl shedding. Furthermore, stimulation of Axl by its ligand, Gas6, induced chemotaxis of human DC and rescued them from growth factor deprivation-induced apoptosis. Our study provides the first evidence that Gas6/Axl-mediated signaling regulates human DC activities, and identifies Gas6/Axl as a new DC chemotaxis pathway. This encourages one to explore whether dysregulation of this novel pathway in human DC biology is involved in autoimmunity characterized by high levels of IFN-alpha.

Monocytes and dendritic cells in a hypoxic environment: Spotlights on chemotaxis and migration.

A common denominator of several pathological conditions, such as solid tumors and inflammatory lesions, is represented by low partial oxygen pressure (pO(2))(2). Mononuclear phagocytes are recruited in large numbers as primary monocytes from the circulation to diseased tissues, where they accumulate within ischemic/hypoxic sites terminally differentiating into inflammatory and tumor-associated macrophages or myeloid dendritic cells (DCs). Thus, mononuclear phagocyte responses that ensue at pathological sites begin in the setting of reduced pO(2). In the last years, extensive work from several groups has been carried out to characterize hypoxia-mediated changes in mononuclear phagocyte gene expression and functional properties under different pathologic situations, demonstrating that oxygen availability is a critical regulator of their functional behavior. However, the majority of reports are focused on the characterization of differentiated macrophages, in particular tumor-infiltrating macrophages (TAM), whereas limited evidence is available for what concerns the responses of peripheral blood monocytes or DCs to the local hypoxic environment. This brief review provides an overview of the phenotypic and functional changes triggered by hypoxia in primary monocytes and DCs. A major focus is given to the chemotactic activity and migratory behavior of these cells when exposed to levels of hypoxia similar to those present in ischemic tissues. Specifically, we discuss the influence of the local hypoxic microenvironment on the expression profile of genes involved in cell motility/migration. Experimental evidence demonstrating that hypoxia modulates in primary monocytes the expression of a selected cluster of chemokine genes with a characteristic dichotomy resulting in the up-regulation of those active on neutrophils and the inhibition of those predominantly active on monocytes, macrophages, T lymphocytes, NK cells, basophils and/or DCs is reported. We also review the findings suggestive of a negative regulatory role of hypoxia on monocyte migration, which is exerted through several alternative or complementary mechanisms and results in monocyte "trapping" within ischemic/hypoxic sites of diseased tissues. Furthermore, we summarize data relative to the ability of hypoxia to differentially regulate in immature DCs (iDCs) the expression profile of genes coding for chemokines and chemokine receptors, the former being down-regulated and the latter up-regulated, thus promoting the switch from a proinflammatory to a migratory phenotype of iDCs by, respectively, reducing their capacity to recruit other inflammatory leukocytes and increasing their sensitivity to chemoattractants. Similarities and differences between the gene expression pattern induced by hypoxia in primary monocytes and that reported in differentiated macrophages are also outlined in this review, to attempt to establish which gene clusters representative of the hypoxic transcriptome of mononuclear phagocytes are specific for a certain stage of differentiation. In particular, we discuss the partial overlap existing among mononuclear phagocytes at various differentiation stages in the expression of a cluster of hypoxia-responsive genes coding for regulators of angiogenesis, proinflammatory cytokines/receptors, and inflammatory mediators and implicated in tissue neo-vascularization and cell activation. Finally, we review studies on the transcription pathways underlying hypoxia-regulated gene expression in monocytic lineage cells, which support a major role for the hypoxia-inducible factor-1 (HIF-1)/hypoxia responsive element (HRE) pathway in monocyte extravasation and migration to hypoxic sites and in the activation of monocyte/macrophage proinflammatory and immunoregulatory responses by hypoxia both in vitro and in vivo. Recent experimental evidence suggesting the requirement of additional transcription factors, such as nuclear factor-kappaB (NF-kappaB), Ets-1, CCAAT/enhancer binding protein-alpha/beta (C/EBPalpha/beta), activator-protein-1 (AP-1), and early growth response-1 (Egr-1), for hypoxic regulation of gene transcription in primary human monocytes and differentiated macrophages and indicative of the existence of both a positive and a negative O(2)-driven HIF-1-dependent feedback regulatory mechanism of hypoxia transcriptional response in primary monocytes, are also reported.

Human dendritic cells differentiated in hypoxia down-modulate antigen uptake and change their chemokine expression profile.

Dendritic cells (DCs) are the most potent antigen-presenting cells and fine-tune the immune response. We have investigated hypoxia's effects on the differentiation and maturation of DCs from human monocytes in vitro, and have shown that it affects DC functions. Hypoxic immature DCs (H-iDCs) significantly fail to capture antigens through down-modulation of the RhoA/Ezrin-Radixin-Moesin pathway and the expression of CD206. Moreover, H-iDCs released higher levels of CXCL1, VEGF, CCL20, CXCL8, and CXCL10 but decreased levels of CCL2 and CCL18, which predict a different ability to recruit neutrophils rather than monocytes and create a proinflammatory and proangiogenic environment. By contrast, hypoxia has no effect on DC maturation. Hypoxic mature DCs display a mature phenotype and activate both allogeneic and specific T cells like normoxic mDCs. This study provides the first demonstration that hypoxia inhibits antigen uptake by DCs and profoundly changes the DC chemokine expression profile and may have a critical role in DC differentiation, adaptation, and activation in inflamed tissues.

Production and function of activin A in human dendritic cells.

Activin A, a member of the transforming growth factor-beta superfamily, has a role in tissue repair and inflammation. In our previous studies, we identified by immunohistochemistry DC-SIGN(+) dendritic cells as a source of activin A in vivo. The present study was aimed at investigating activin A production by dendritic cells (DC) in vitro and its function. Here we demonstrate that monocyte-derived DC (Mo-DC) released abundant levels of activin A during the maturation process induced by TLR agonists, bacteria (B. henselae, S. thyphimurium), TNF and CD40L. Activin A was also induced in monocyte-derived Langerhans cells (LC) and in blood myeloid DC by LPS and/or CD40L stimulation, but not in blood plasmacytoid DC following stimulation with influenza virus. Activin A production by DC was selectively down-regulated by anti-inflammatory molecules such as dexamethasone or IL-10. Neutralization of endogenous activin A using its inhibitor follistatin, or the addition of exogenous activin A during LPS maturation did not affect Mo-DC maturation marker expression, cytokine release or allostimulatory function. However, Mo-DC matured with LPS in the presence of exogenous activin A displayed a higher FITC-dextran uptake, similar to that of immature DC. Moreover, activin A promoted monocyte differentiation to DC and reversed the inhibitory effects of IL-6 on DC differentiation of monocytes. These findings demonstrate that different subsets of DC release activin A, a cytokine that promotes DC generation, and affects the ability of mature DC to take up antigens (Ags).

CC-chemokine ligand 16 induces a novel maturation program in human immature monocyte-derived dendritic cells.

Dendritic cells (DCs) are indispensable for initiation of primary T cell responses and a host's defense against infection. Many proinflammatory stimuli induce DCs to mature (mDCs), but little is known about the ability of chemokines to modulate their maturation. In the present study, we report that CCL16 is a potent maturation factor for monocyte-derived DCs (MoDCs) through differential use of its four receptors and an indirect regulator of Th cell differentiation. MoDCs induced to mature by CCL16 are characterized by increased expression of CD80 and CD86, MHC class II molecules, and ex novo expression of CD83 and CCR7. They produce many chemokines to attract monocytes and T cells and are also strong stimulators in activating allogeneic T cells to skew toward Th1 differentiation. Interestingly, they are still able to take up Ag and express chemokine receptors usually bound by inflammatory ligands and can be induced to migrate to different sites where they capture Ags. Our findings indicate that induction of MoDC maturation is an important property of CCL16 and suggest that chemokines may not only organize the migration of MoDCs, but also directly regulate their ability to prime T cell responses.