Albumin-binding recombinant human IL-18BP ameliorates macrophage activation syndrome and atopic dermatitis via direct IL-18 inactivation.

Given the clinical success of cytokine blockade in managing diverse inflammatory human conditions, this approach could be exploited for numerous refractory or uncontrolled inflammatory conditions by identifying novel targets for functional blockade. Interleukin (IL)-18, a pro-inflammatory cytokine, is relatively underestimated as a therapeutic target, despite accumulated evidence indicating the unique roles of IL-18 in acute and chronic inflammatory conditions, such as macrophage activation syndrome. Herein, we designed a new form of IL-18 blockade, i.e., APB-R3, a long-acting recombinant human IL-18BP linked to human albumin-binding Fab fragment, SL335, for extending half-life. We then explored the pharmacokinetics and pharmacodynamics of APB-R3. In addition to an extended serum half-life, APB-R3 alleviates liver inflammation and splenomegaly in a model of the macrophage activation syndrome induced in IL-18BP knockout mice. Moreover, APB-R3 substantially controlled skin inflammation in a model of atopic dermatitis. Thus, we report APB-R3 as a new potent IL-18 blocking agent that could be applied to treat IL-18-mediated inflammatory diseases.

Long-acting recombinant human follicle-stimulating hormone (SAFA-FSH) enhances spermatogenesis.

Introduction: Administration of follicle-stimulating hormone (FSH) has been recommended to stimulate spermatogenesis in infertile men with hypogonadotropic hypogonadism, whose sperm counts do not respond to human chorionic gonadotropin alone. However, FSH has a short serum half-life requiring frequent administration to maintain its therapeutic efficacy. To improve its pharmacokinetic properties, we developed a unique albumin-binder technology, termed "anti-serum albumin Fab-associated" (SAFA) technology. We tested the feasibility of applying SAFA technology to create long-acting FSH as a therapeutic candidate for patients with hypogonadotropic hypogonadism. Methods: SAFA-FSH was produced using a Chinese hamster ovary expression system. To confirm the biological function, the production of cyclic AMP and phosphorylation of ERK and CREB were measured in TM4-FSHR cells. The effect of gonadotropin-releasing hormone agonists on spermatogenesis in a hypogonadal rat model was investigated. Results: In in vitro experiments, SAFA-FSH treatment increased the production of cyclic AMP and increased the phosphorylation of ERK and CREB in a dose-dependent manner. In animal experiments, sperm production was not restored by human chorionic gonadotropin treatment alone, but was restored after additional recombinant FSH treatment thrice per week or once every 5 days. Sperm production was restored even after additional SAFA-FSH treatment at intervals of once every 5 or 10 days. Discussion: Long-acting FSH with bioactivity was successfully created using SAFA technology. These data support further development of SAFA-FSH in a clinical setting, potentially representing an important advancement in the treatment of patients with hypogonadotropic hypogonadism.

Expression of soluble and functional human neonatal Fc receptor in Pichia pastoris.

The neonatal Fc receptor (FcRn) is a non-covalently associated heterodimeric protein composed of a transmembrane anchored heavy chain (alphaFcRn) and a soluble light chain beta2-microglobulin (beta2m). In addition to its role in the transfer of maternal immunoglobulin Gs (IgGs) to the fetus, FcRn plays a key role in prolonging the serum half-life of IgGs in vivo. Herein, we report a strategy for functional expression of soluble human FcRn (shFcRn) in Pichia pastoris using a two-promoter vector system, where alphaFcRn and beta2m are co-expressed under their respective promoters in a single vector. The purified shFcRn from the culture supernatants correctly assembled to form the heterodimer with the typical secondary structures. At acidic pHs between 5.0 and 6.4, shFcRn exhibited substantial binding to the four subclasses of human IgGs at acidic pHs between 5.0 and 6.4, but at pHs between 6.8 and 8.0, its binding was negligible binding. No cross-reactivity with mouse IgG was exhibited even at acidic pH. This was consistent with the pH-dependent binding profiles of the shFcRn prepared from the mammalian cell expression. Furthermore, the shFcRn exhibited about 10-fold higher binding affinity with the tumor necrosis factor-alpha antagonists of monoclonal antibodies Infliximab and Adalimumab than that of Etanercept, providing a clue to their different serum half-lives in vivo. Our results suggest that the functionally expressed shFcRn from Pichia can be used for the biochemical and biological studies and as a screening probe for Fc engineering of human IgGs.

Characterization of a novel anti-human TNF-alpha murine monoclonal antibody with high binding affinity and neutralizing activity.

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta. TSK114 was found to be of IgG1 isotype with kappa light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-alpha by surface plasmon resonance technique revealed a binding affinity (K(D)) of approximately 5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, respectively. TSK114 neutralized human TNF-alpha-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-alpha-neutralizing antibody with picomolar affinity.

Comparative analyses of complex formation and binding sites between human tumor necrosis factor-alpha and its three antagonists elucidate their different neutralizing mechanisms.

Tumor necrosis factor-alpha (TNFalpha)-blocking therapy, using biologic TNFalpha antagonists, has been approved for the treatment of several diseases including rheumatoid arthritis, psoriasis and Crohn's disease. There have been few detailed studies of binding characterizations for the complex formation by TNFalpha and clinically relevant antagonists, particularly Infliximab (Remicade) and Etanercept (Enbrel). Here we characterized the binding stoichiometry and size of soluble TNFalpha-antagonist complexes and identified energetically important binding sites on TNFalpha for the three antagonists, Etanercept, Infliximab, and the recently developed humanized TNFalpha neutralizing monoclonal antibody, YHB1411-2. Size-exclusion chromatography and dynamic light scattering analyses revealed that the three antagonists formed distinct thermodynamically stable TNFalpha-antagonist complexes that exhibited differences in their size and composition. Energetically important binding residues on TNFalpha were identified for each antagonist by a sequence of experiments that consisted of competition binding assays, fragmentations, loop mutations, and single-point mutations using yeast surface-displayed TNFalpha, which was further confirmed for solubly purified TNFalpha mutants by surface plasmon resonance technique. Analyses of the binding geometry based on binding site location, spatial constraints, and valency satisfaction allowed us to interpret the thermodynamically stable complexes as follows: one molecule of Etanercept and one molecule of trimeric TNFalpha (Etanercept1-TNFalpha1), Infliximab6-TNFalpha3, and YHB1411-2(4)-TNFalpha2. The distinct features of the soluble antagonist-TNFalpha complex formation among the antagonists may give further insights into their different neutralizing mechanisms and pharmacokinetic profiles.