Generic approach for the generation of stable humanized single-chain Fv fragments from rabbit monoclonal antibodies.

Despite their favorable pharmacokinetic properties, single-chain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 x 10(-9) to 1.5 x 10(-11) m. Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 x 10(-10) to <1.8 x 10(-12) m. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86 degrees C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains.

Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments.

Multispecific antibody formats provide a promising platform for the development of novel therapeutic concepts that could facilitate the generation of safer, more effective pharmaceuticals. However, the production and use of such antibody-based multispecifics is often made complicated by: 1) the instability of the antibody fragments of which they consist, 2) undesired inter-subunit associations, and 3) the need to include recombinant heterodimerization domains that confer distribution-impairing bulk or enhance immunogenicity. In this paper, we describe a broadly-applicable method for the stabilization of human or humanized antibody Fv fragments that entails replacing framework region IV of a Vκ1/VH3-consensus Fv framework with the corresponding germ-line sequence of a λ-type VL chain. We then used this stable Fv framework to generate a novel heterodimeric multispecific antibody format that assembles by cognate VL/VH associations between 2 split variable domains in the core of the complex. This format, termed multispecific antibody-based therapeutics by cognate heterodimerization (MATCH), can be applied to produce homogeneous and highly stable antibody-derived molecules that simultaneously bind 4 distinct antigens. The heterodimeric design of the MATCH format allows efficient in-format screening of binding domain combinations that result in maximal cooperative activity.

Cell growth selection system to detect extracellular and transmembrane protein interactions.

The interplay among extracellular and cell surface proteins, such as the interactions between ligands and receptors or between antigens and antibodies, is involved in a multitude of physiological and pathological phenomena. In the oxidizing milieu of the secretory pathway in eukaryotic cells, many extracellular proteins build disulfide bonds that significantly contribute to their correct folding and structural stability. Thus, conventional yeast two-hybrid interaction assays, which occur in the reducing intracellular environment, might not be adequate to detect extracellular protein-protein interactions. We have exploited the properties of yeast Ire1p, a type I endoplasmic reticulum (ER) membrane protein involved in the unfolded protein response (UPR) as a dimerization-activated receptor, to develop a novel system for the detection and study of interactions between extracellular and/or membrane proteins. In our system, named SCINEX-P (screening for interactions between extracellular proteins), proteins of interest were fused to truncated Ire1p so as to substitute its N-terminal lumenal domain (NLD). Specific interaction between two partners caused dimerization of the Ire1p moiety, which, through the endogenous UPR signalling pathway, led to activation of transcription of genes that permit cell growth under selective conditions.

Injectable formulations for an intravitreal sustained-release application of a novel single-chain VEGF antibody fragment.

Sustained-release formulations of a single-chain anti-VEGF-A antibody fragment were investigated in vitro toward their potential use for intravitreal applications. The hydrophobic polyester hexylsubstituted poly(lactic acid) (hexPLA) was selected as the sustained-release excipient for its biodegradability and semi-solid aggregate state, allowing an easy and mild formulation procedure. The lyophilized antibody fragment ESBA903 was micronized and incorporated into the liquid polymer matrix by cryo-milling, forming homogeneous and injectable suspensions. The protein showed excellent compatibility with the hexPLA polymer and storage stability at 4°C for 10 weeks. Additionally, hexPLA shielded the incorporated active substance from the surrounding medium, resulting in a better stability of ESBA903 inside the polymer than after its release in the buffer solution. Formulations of ESBA903 with hexPLA having drug loadings between 1.25% and 5.0% and polymer molecular weights of 1500 g/mol, 2500 g/mol, 3500 g/mol and 5000 g/mol were investigated regarding their in vitro release. All formulations except with the highest molecular weight formed spherical depots in aqueous buffer solutions and released the antibody fragment for at least 6-14 weeks. The polymer viscosity derived from the molecular weight strongly influenced the release rate, while the drug loading had minor influence, allowing customization of the release profile and the daily drug release. Size exclusion chromatography and SDS-PAGE revealed that the antibody fragment structure was kept intact during incorporation and release from the liquid matrix. Furthermore, the released protein monomer maintained its high affinity to human VEGF-A, as measured by surface plasmon resonance analysis. Formulations of ESBA903 in hexPLA meet the basic needs to be used for intravitreal sustained-release applications in age-related macular degeneration treatment.

Relative contribution of VEGF and TNF-alpha in the cynomolgus laser-induced CNV model: comparing the efficacy of bevacizumab, adalimumab, and ESBA105.

PURPOSE: To compare the relative contribution of VEGF and TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in monkeys and to exploit the feasibility of topical use of suitable antibody fragments for the prevention of experimental CNV. METHODS: To induce experimental CNV, small high-energy laser spots were used to treat several areas of the macula in the retinas of cynomolgus monkeys according to previously published protocols. To prevent abnormalities, bevacizumab (a potent VEGF inhibitor) and adalimumab or ESBA105 (potent TNF-alpha inhibitors) were given by intravitreal injection 1 week before and 1 week and 3 weeks after laser treatment. ESBA105 was also applied topically in a separate group. Control animals were treated with either intravitreal or topical saline. Eyes were monitored by ophthalmic examination, color photography, and fluorescein angiography. RESULTS: Inhibition of VEGF by bevacizumab completely blocked the formation of CNV. Both TNF-alpha inhibitors also significantly reduced laser-induced CNV abnormalities after intravitreal administration. Most important, topical use of the anti-TNF-alpha single-chain antibody fragment ESBA105 also reduced the formation of CNV. CONCLUSIONS: TNF-alpha contributes to laser-induced CNV formation, and its inhibition can be a new therapeutic target for CNV. This study suggests TNF-alpha as another therapeutic target for the prevention and treatment of CNV and adds to the emerging clinical data suggesting the therapeutic value of TNF-alpha inhibitors in age-related macular degeneration (AMD). Further, this study shows that topical therapy with suitable antibody fragments has the potential of being introduced to retinal disease treatment regimens.

Intranasal delivery of ESBA105, a TNF-alpha-inhibitory scFv antibody fragment to the brain.

Intranasal drug administration is an attractive route for targeted delivery of large molecular weight compounds to the central nervous system (CNS). The purpose of this study was to assess the feasibility of this non-invasive application method in mice, for delivery of ESBA105, a TNF-alpha inhibitory single-chain antibody fragment (scFv) with a molecular weight of 26.3kDa, to the brain. Pharmacokinetic parameters were determined for different brain regions (olfactory bulb, cerebrum, cerebellum, brain stem) and for serum, following both, intranasal and intravenous administrations of 400microg and 40microg ESBA105, respectively. ESBA105 efficiently migrated from the nasal cavity to the brain and maximum ESBA105 concentrations (C(max)) in the brain were measured between 1.1 and 12.2microg/mg of the total protein. Although a 10-fold higher dose was given intranasally, systemic exposure was about 33-fold lower for the intranasal route than following systemic application. Addition of a penetration enhancing peptide to the formulation enhanced the delivery of ESBA105 to the olfactory bulb and the cerebrum, without increasing systemic exposure.

Efficient intraocular penetration of topical anti-TNF-alpha single-chain antibody (ESBA105) to anterior and posterior segment without penetration enhancer.

PURPOSE: This study was designed to characterize ocular penetration pathways of ESBA105, a topically administered single-chain antibody (scFv) against tumor necrosis factor (TNF)-alpha, to the anterior and posterior segment of the eye. METHODS: Fresh enucleated whole eyes and isolated corneas of rabbits mounted in perfusion chambers were used for ex vivo penetration studies. In vivo pharmacokinetics and ocular biodistribution of ESBA105 after intravitreal injection or topical administration as eye drops were investigated in rabbits. RESULTS: After topical administration as eye drops, without a penetration enhancer, ESBA105 reached therapeutic levels in the anterior and posterior segment of the eye. ESBA105 migrated to aqueous humor via corneal penetration and vitreous and retina via intrascleral penetration pathways. In vivo, ESBA105 had a significantly prolonged elimination half-life in the vitreous of 25 hours compared with its serum half-life of 7 hours after i.v. administration. Therefore, based on frequency of topical dosing, a buildup of ESBA105 to distinct steady state levels in the vitreous could be achieved. CONCLUSIONS: Topically administered ESBA105 quickly reaches therapeutic levels in the anterior and posterior segment without any need for a penetration enhancer. Drug penetration and ocular biodistribution patterns of ESBA105 applied as eye drops appear highly attractive for clinical use to treat TNF-alpha dependant diseases of the eye.

Pharmacokinetics and posterior segment biodistribution of ESBA105, an anti-TNF-alpha single-chain antibody, upon topical administration to the rabbit eye.

PURPOSE: The purpose of this study was to characterize local distribution and systemic absorption of the tumor necrosis factor (TNF)-alpha inhibitory single-chain antibody fragment (scFv) ESBA105 following topical administration to the eye in vivo. METHODS: Rabbits received ESBA105 as topical eye drops in two dosing regimens. First, pharmacokinetics after the topical route of administration was compared to the intravenous (i.v.) route by means of applying the identical cumulative daily dose of ESBA105. In a second study rabbits received five eye drops daily for six consecutive days in a lower frequency topical dosing regimen. Kinetics and biodistribution of ESBA105 in ocular tissues and fluids as well as in sera were determined in all animals. RESULTS: After topical administration to the eye, ESBA105 quickly reaches therapeutic concentrations in all ocular compartments. Systemic exposure after topical administration is 25,000-fold lower than exposure after i.v. injection of the identical cumulative daily dose. ESBA105 levels in vitreous humor and neuroretina are significantly higher on topical administration than after i.v. injection. Absolute and relative intraocular biodistribution of ESBA105 is different with topical and systemic delivery routes. Compared to its terminal half-life in circulation (7 hours), the vitreal half-life of ESBA105 is significantly enhanced (16-24 hours). CONCLUSIONS: On topical administration, ESBA105 is efficiently absorbed and distributed to all compartments of the eye, whereby systemic drug exposure is very low. Based on its unique intraocular biodistribution and pharmacokinetics and the absolute intraocular levels reached, topical ESBA105 appears highly attractive for treatment of various ophthalmological disorders.