Effectiveness of Soluble CTLA-4-Fc in the Inhibition of Bone Marrow T-Cell Activation in Context of Indoleamine 2.3-Dioxygenase (IDO) and CD4+Foxp3+ Treg Induction.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease with systemic inflammation finally resulting in damaged joints. One of the RA development models suggests bone marrow (BM) as a place of inflammation development further leading to disease progression. We aimed to investigate the potential of CTLA-4-Fc molecule in inducing tolerogenic milieu in BM measured as indoleamine 2,3-dioxygenase (IDO) expression, CD4+Foxp3+ Treg induction, and T cell activation control. The expression of IDO-pathway genes was also examined in monocytes to estimate the tolerogenic potential in the periphery. Methods: Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4+CD69+ and CD4+Foxp3+ percentage were estimated by PCR and FACS staining, respectively. Enzymatic activity of IDO was confirmed by HPLC in BM plasma and blood plasma. Genes expressed in IDO-pathway were analyzed by NGS in peripheral monocytes isolated from RA patients and healthy controls. Results: We found that CTLA-4-Fc and IFN-γ stimulation results in IDO production by BMMC. CTLA-4-Fc induced tryptophan catabolism can inhibit mitogen-induced CD4+ T cells activation without influencing CD8+ cells, but did not control CD25 nor Foxp3 expression in BM cells. Significantly higher expression of selected IDO-pathway genes was detected on peripheral monocytes isolated from RA as compared to healthy controls. Conclusion: This study sheds light on some immunosuppression aspects present or induced in BM. The potential of IDO-mediated pathways were confirmed in the periphery, what may represent the promising candidates for therapeutic strategies in RA.

Abatacept (Cytotoxic T Lymphocyte Antigen 4-Fragment Crystallizable) Reduces Allergic Inflammation of Ovalbumin-Sensitized Mice.

BACKGROUND: Abatacept (Aba) is a cytotoxic T-lymphocyte antigen-4 and fragment crystallizable fusion protein. Aba blocks B7/Cluster of differentiation 28 - cytotoxic T-lymphocyte antigen-4 costimulatory pathway, inhibits cluster of differentiation 4+ T-cell activation, and is used as an anti-inflammatory drug. OBJECTIVES: We conducted this study to assess the effectiveness of Aba in the treatment of allergic rhinitis (AR) in a mouse model. METHODS: We divided 40 four-week-old BALB/c mice into four groups: control group (n = 10), positive control group (AR, n = 10), Aba group (AR + Aba, n = 10), and dexamethasone group (AR + Dex, n = 10). Mice in each group were challenged intranasally with daily ovalbumin (OVA) administration. Episodes of sneezing and nose rubbing were counted. Mice were sacrificed on day 42 and cytokines were measured in nasal lavage fluid. Nasal mucosae of five mice from each group were used for reverse transcriptase-polymerase chain reaction and western blot assay. Samples were collected from five mice from each group for histological analysis. RESULTS: Symptoms of AR significantly improved in the AR + Aba and AR + Dex groups compared with the AR group. Fewer eosinophils and goblet cells were seen in the AR + Aba and AR + Dex groups compared with the AR group. Both the AR + Aba and AR + Dex groups showed a significant decrease in nasal T helper 2 cytokine levels, including interleukin (IL)-4, IL-5, IL-13 and T cell activation related IL-17A, and interferon gamma (IFN- γ). Total immunoglobulin (Ig) E and OVA-specific IgG1 levels were also significantly lower in the AR + Aba and AR + Dex groups. OVA-specific IgE level was also significantly lower in the AR + Aba than AR group. CONCLUSIONS: Aba suppresses allergic inflammation and appears to be a good treatment for AR.

Abatacept for treatment-refractory pediatric CTLA4-haploinsufficiency.

CTLA4-haploinsufficiency is a complex disease of immune dysregulation presenting with a broad spectrum of clinical manifestations. CTLA4-Fc fusion proteins such as abatacept have been described to alleviate immune dysregulation in several adult cases of CTLA4-haploinsufficiency. However, until now only few cases of pediatric CTLA4-haploinsufficiency treated with abatacept have been described. Here we present two pediatric cases of severe CTLA4-haploinsufficiency refractory to conventional immunosuppressive therapies that responded rapidly to treatment with abatacept. No side effects were observed during a follow-up period of 7-15 months. While one patient has successfully undergone HSCT the second patient continues to receive abatacept. Our cases demonstrate safe medium-term use of abatacept in the pediatric population.

Site-specific monitoring of N-Glycosylation profiles of a CTLA4-Fc-fusion protein from the secretory pathway to the extracellular environment.

Glycosylation often plays a key role in the safety and efficacy of therapeutic proteins to patients, thus underlying the need for consistent control of this important post-translational modification during biologics production. In this study, we profiled the site-specific evolution of N-glycans on a CTLA4-Fc-fusion protein, from the intracellular secretory pathway to the conditioned medium (CM) in fed-batch cell culture. For this, we developed an approach that combined sub-cellular fractionation with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The study revealed that there was a significant amount of heterogeneity in the glycans displayed amongst the three distinct N-glycosylation sites. Furthermore, 54-60% of the intracellular protein was characterized by Man8 and Man9 glycans on day 10, when the cell density peaks, indicative of a significant bottleneck between the endoplasmic reticulum (ER) and the cis-Golgi. At longer culture duration, the accumulation of intracellular protein with bi-antennary-fucosylated GlcNAc-terminated residues identified the formation of another bottleneck in the medial and trans-Golgi compartments, which subsequently led to a decrease in sialylated species in the secreted protein. Glucose deprivation caused a reduction in the Man8 and Man9 glycans in favor of Man5 glycans and bi-antennary-fucosylated GlcNAc-terminated residues in the organellar pool of the Fc-fusion protein. However, transient deprivation of glucose did not lead to major differences in the glycan profile of proteins secreted into the CM. The approach developed here allows us to probe the secretory pathway and sheds light on the site-specific intracellular processing of glycans during fed-batch cell culture, thus serving as an initial step towards their rational control. Biotechnol. Bioeng. 2017;114: 1550-1560. © 2017 Wiley Periodicals, Inc.

Inhibition of T-cell activation by the CTLA4-Fc Abatacept is sufficient to ameliorate proteinuric kidney disease.

Diabetic nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T-cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T-cell activation will ameliorate DN. Interaction of B7-1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T-cells, is essential for T-cell activation. In this study we used the soluble CTLA4-Fc fusion protein Abatacept to block cell surface B7-1, preventing the cellular interaction and inhibiting T-cell activation. When Abatacept was dosed in an animal model of diabetes-induced albuminuria, it reduced albuminuria in both prevention and intervention modes. The number of T-cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria, and treatment with Abatacept reduced the number of renal T-cells. As B7-1 induction has been recently proposed to underlie podocyte damage in DN, Abatacept could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7-1 was not expressed in 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients. We conclude that Abatacept ameliorates DN by blocking systemic T-cell activation and not by interacting with podocytes.