Frank Kolbinger

Effect of IL-17A blockade with secukinumab in autoimmune diseases

Genetic studies and correlative expression data in diseased tissues have pointed to the role of interleukin (IL)-17 and Th17 cells in the pathogenesis of autoimmune disorders such as psoriasis, inflammatory bowel disease and seronegative spondyloarthropathies. Th17 cells are known to produce the proinflammatory cytokine IL-17A as well as other effector cytokines, including IL-17F and IL-22. Recent research has demonstrated that IL-17A is also expressed by multiple lineages of the innate immune system, including mast cells, neutrophils, dendritic cells, γδ-T cells, macrophages and natural killer cells. It can thus be expected that the inhibition of IL-17A as a therapeutic target in autoimmune disease would exert different physiological effects than the suppression of Th17 cell activity. Early clinical data are now available on secukinumab (AIN457), a recombinant, highly selective, fully human monoclonal anti-IL-17A antibody of the IgG1/κ isotype, enabling a preliminary assessment of the effects of IL-17A inhibition in multiple autoimmune diseases. Rapid and sustained symptom reductions in psoriasis, rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis have been observed in secukinumab-treated patients, with no overt safety signals. In conjunction with studies using the humanised anti-IL-17A monoclonal antibody (mAb) ixekizumab (LY2439821) and the fully human anti-IL-17RA mAb brodalumab (AMG 827), the findings on secukinumab provide evidence for the role of IL-17A in the pathophysiology of autoimmune disease and suggest the potential value of targeting this cytokine.

Evidence that a neutrophil–keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis

The response of psoriasis to antibodies targeting the interleukin (IL)‐23/IL‐17A pathway suggests a prominent role of T‐helper type‐17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate‐to‐severe psoriasis receiving 3 different intravenous dosing regimens of the anti‐IL‐17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL‐17 and may store the cytokine preformed, as IL‐17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL‐17‐inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c‐positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest‐dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil–keratinocyte axis in psoriasis that may involve neutrophil‐derived IL‐17 and is an early target of IL‐17A‐directed therapies such as secukinumab.

CLONING OF A HUMAN UDP-GALACTOSE :2-ACETAMIDO-2-DEOXY-D-GLUCOSE 3BETA -GALACTOSYLTRANSFERASE CATALYZING THE FORMATION OF TYPE 1 CHAINS

Biochemical evidence suggests that the galactosyltransferase activity synthesizing type 1 carbohydrate chains is separate from the well characterized enzyme that is responsible for the synthesis of type 2 chains. This was recently confirmed by the cloning, from melanoma cells, of an enzyme capable of synthesizing type 1 chains, which was shown to have no homology to other galactosyltransferases. We report here the molecular cloning and functional expression of a second human β3-galactosyltransferase distinct from the melanoma enzyme. The new β3-galactosyltransferase has homology to the melanoma enzyme in the putative catalytic domain, but has longer cytoplasmic and stem regions and a carboxyl-terminal extension. Northern blots showed that the new gene is present primarily in brain and heart. When transfected into mammalian cells, this gene directs the synthesis of type 1 chains as determined by a monoclonal antibody specific for sialyl Lewisa. A soluble version of the cloned enzyme was expressed in insect cells and purified. The soluble enzyme readily catalyzes the transfer of galactose to GlcNAc to form Gal(β1–3)GlcNAc. It also has a minor but distinct transfer activity toward Gal, LacNAc, and lactose, but is inactive toward GalNAc.

Systematic identification of regulatory proteins critical for T-cell activation

Background The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. Results In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10Rα and integrin α2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. Conclusions This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.

Different Adaptations of IgG Effector Function in Human and Nonhuman Primates and Implications for Therapeutic Antibody Treatment

Safety of human therapeutic Abs is generally assessed in nonhuman primates. Whereas IgG1 shows identical FcγR interaction and effector function profile in both species, fundamental differences in the IgG2 and IgG4 Ab subclasses were found between the two species. Granulocytes, the main effector cells against IgG2- and IgG4-opsonized bacteria and parasites, do not express FcγRIIIb, but show higher levels of FcγRII in cynomolgus monkey. In humans, IgG2 and IgG4 adapted a silent Fc region with weak binding to FcγR and effector functions, whereas, in contrast, cynomolgus monkey IgG2 and IgG4 display strong effector function as well as differences in IgG4 Fab arm exchange. To balance this shift toward activation, the cynomolgus inhibitory FcγRIIb shows strongly increased affinity for IgG2. In view of these findings, in vitro and in vivo results for human IgG2 and IgG4 obtained in the cynomolgus monkey have to be cautiously interpreted, whereas effector function-related effects of human IgG1 Abs are expected to be predictable for humans.

An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells

The effects of a chimeric monoclonal antibody (chA6 mAb) that recognizes both the RO and RB isoforms of the transmembrane protein tyrosine phosphatase CD45 on human T cells were investigated. Chimeric A6 (chA6) mAb potently inhibited antigen-specific and polyclonal T cell responses. ChA6 mAb induced activation-independent apoptosis in CD4+CD45RO/RBhigh T cells but not in CD8+ T cells. In addition, CD4+ T cell lines specific for tetanus toxoid (TT) generated in the presence of chA6 mAb were anergic and suppressed the proliferation and interferon (IFN)-γ production by TT-specific effector T cells by an interleukin-10–dependent mechanism, indicating that these cells were equivalent to type 1 regulatory T cells. Similarly, CD8+ T cell lines specific for the influenza A matrix protein-derived peptide (MP.58-66) generated in the presence of chA6 mAb were anergic and suppressed IFN-γ production by MP.58-66–specific effector CD8+ T cells. Furthermore, chA6 mAb significantly prolonged human pancreatic islet allograft survival in nonobese diabetic/severe combined immunodeficiency mice injected with human peripheral blood lymphocytes (hu-PBL-NOD/SCID). Together, these results demonstrate that the chA6 mAb is a new immunomodulatory agent with multiple modes of action, including deletion of preexisting memory and recently activated T cells and induction of anergic CD4+ and CD8+ regulatory T cells.

Secukinumab, a novel anti–IL-17A antibody, shows low immunogenicity potential in human in vitro assays comparable to other marketed biotherapeutics with low clinical immunogenicity

ABSTRACT Secukinumab is a human monoclonal antibody that selectively targets interleukin-17A and has been demonstrated to be highly efficacious in the treatment of moderate to severe plaque psoriasis, starting at early time points, with a sustained effect and a favorable safety profile. Biotherapeutics—including monoclonal antibodies (mAbs)—can be immunogenic, leading to formation of anti-drug antibodies (ADAs) that can result in unwanted effects, including hypersensitivity reactions or compromised therapeutic efficacy. To gain insight into possible explanations for the clinically observed low immunogenicity of secukinumab, we evaluated its immunogenicity potential by applying 2 different in vitro assays: T-cell activation and major histocompatibility complex–associated peptide proteomics (MAPPs). For both assays, monocyte-derived dendritic cells (DCs) from healthy donors were exposed in vitro to biotherapeutic proteins. DCs naturally process proteins and present the derived peptides in the context of human leukocyte antigen (HLA)-class II. HLA-DR–associated biotherapeutic-derived peptides, representing potential T–cell epitopes, were identified in the MAPPs assay. In the T-cell assay, autologous CD4+ T cells were co-cultured with secukinumab-exposed DCs and T-cell activation was measured by proliferation and interleukin-2 secretion. In the MAPPs analysis and T-cell activation assays, secukinumab consistently showed relatively low numbers of potential T-cell epitopes and low T-cell response rates, respectively, comparable to other biotherapeutics with known low clinical immunogenicity. In contrast, biotherapeutics with elevated clinical immunogenicity rates showed increased numbers of potential T-cell epitopes and increased T-cell response rates in T-cell activation assays, indicating an approximate correlation between in vitro assay results and clinical immunogenicity incidence.

β-Defensin 2 is a responsive biomarker of IL-17A-driven skin pathology in patients with psoriasis.

Background: IL‐17A is a key driver of human autoimmune diseases, particularly psoriasis. Objective: We sought to determine the role of IL‐17A in psoriasis pathogenesis and to identify a robust and measurable biomarker of IL‐17A–driven pathology. Methods: We studied 8 healthy subjects and 8 patients with psoriasis before and after administration of secukinumab, a fully human anti–IL‐17A mAb, and used a combination of classical techniques and a novel skin microperfusion assay to evaluate the expression of 170 proteins in blood, nonlesional skin, and lesional skin. For validation, we also tested stored sera from 601 patients with a variety of autoimmune diseases. Results: IL‐17A was specifically expressed in lesional compared with nonlesional psoriatic skin (9.8 vs 0.8 pg/mL, P < .001). Proteomic and gene transcription analyses revealed dysregulated antimicrobial peptides, proinflammatory cytokines, and neutrophil chemoattractants, levels of which returned to normal after treatment with secukinumab. &bgr;‐Defensin 2 (BD‐2) was identified as a biomarker of IL‐17A–driven pathology by comparing protein expression in patients with psoriasis versus that in healthy subjects (5746 vs 82 pg/mL in serum, P < .0001; 2747 vs <218 pg/mL in dermis, P < .001), responsiveness to secukinumab therapy, and synergistic induction by IL‐17A and TNF‐&agr; in epidermal keratinocytes. In a validation set of sera from 601 patients with autoimmune diseases thought to be IL‐17A driven, we found that BD‐2 levels are most highly increased in patients with psoriatic skin lesions, and in patients with psoriasis, BD‐2 levels correlated well with IL‐17A levels (r = 0.70, n = 199, P < .001) and Psoriasis Area and Severity Index scores (r = 0.53, n = 281, P < .001). Conclusion: IL‐17A is a primary driver of skin pathology in patients with psoriasis, and serum BD‐2 is an easily measurable biomarker of IL‐17A–driven skin pathology.

Synthetic potential of cloned fucosyl-transferase III and VI

Abstract Two cloned fucosyl-transferases, fuc-t III and fuc-t VI, are probed with non-natural donor-sugars, in which the natural fucose moiety is replaced by mono-saccharides modified in their 2- or 6-positions. Despite their close sequence homology, the investigated transferases exhibit significant differences in their substrate recognition.

Automated baculovirus titration assay based on viable cell growth monitoring using a colorimetric indicator

The fastest methods reported are based on viral DNA quantitation using either flow cytometry (10) or real-time PCR (11), which allow titer determination within two hours. The drawbacks of these approaches are the costs of equipment and staining reagents, and the fact that the total number of particles and not the number of infectious particles is determined. An alternative approach is based on the lytic nature of the viral system (12), and more specifically on the fact that cell growth is attenuated upon virus infection. This growth reduction is dose-dependent and can be estimated by measuring the viable cell concentration and subsequently correlating this to the virus titer. Indeed, a new method was recently developed for virus titration by spectrophotometrically monitoring the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) (4). The accuracy of the method was clearly demonstrated; however, the number of preparation steps and the overall duration (6 days) are not compatible with a fast and automated high-throughput process. In this work, we demonstrate that