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Intact Bioactivities and Improved Pharmacokinetic of the SL335-IFN-β-1a Fusion Protein that Created by Genetic Fusion of SL335, a Human Anti-serum Albumin Fab, and Human Interferon-β
Abstract
Recombinant human interferon beta (rIFN-β) has long been used as a first-line treatment for multiple sclerosis (MS), and any attempt to develop a long-acting rIFN-β is desirable since only one pegylated version of long-acting rIFN-β-1a (Plegridy) is currently available in clinics. Previously, we reported that SL335, a human Fab molecule specific to serum albumin, exhibits an extended serum half-life via utilizing the FcRn recycling mechanism. With the ultimate goal of developing a long-acting rIFN-® we generated a fusion construct by linking human IFN-β cDNA to the C-terminus of the SL335 H chain at the DNA level followed by expression of the fusion protein, referred to as SL335-IFN-β-1a, in Chinese hamster ovary-S (CHO-S) cells. In its N-linked glycosylated form, the resulting fusion protein was easily purified from the culture supernatant via a three-step chromatography process. In vitro functional assays revealed that the fusion protein retained its intrinsic binding capabilities to human serum albumin (HSA) and interferon α/β receptor (IFNAR) that were almost identical to those of parental SL335 and rIFN-β-1a (Rebif). In addition, the fusion protein possessed an antiviral potency and anti-proliferation activity comparable to those of Rebif. In pharmacokinetic (PK) analyses using Lewis rats and cynomolgus monkeys, SL335-IFN-β-1a exhibited at least a two-fold longer serum half-life and a significantly reduced renal clearance rate compared to those of Rebif. Finally, a four-week repeated dose toxicity study revealed no abnormal toxicological signs. In conclusion, our results clearly demonstrated that SL335-IFN-β-1a is worthy of further development as an alternative long-acting IFN-β therapeutic.
... We previously developed a unique albumin-binder technology that profoundly extended the half-life of a therapeutic protein, termed "anti-serum albumin Fab-associated" (SAFA) technology (12,13). Using this technology, a long-acting recombinant human interferon beta (at least 2-fold longer serum half-life in rats and monkeys) for multiple sclerosis and a long-acting feline granulocyte colony-stimulating factor (approximately 5-fold longer serum halflife in cats) were developed (14,15). In the present study, we tested the feasibility of applying SAFA technology to develop long-acting FSH as a therapeutic candidate for patients with HH. ...
... 2 Materials and methods 2.1 SAFA-FSH production and purification SAFA-FSH was produced by CHO glutamine synthetase null −/− K1 cell (Horizon Discovery, Waterbeach, UK) and purified using a three step purification protocol-capture for affinity chromatography, intermediate purification for multimodal chromatography, and polishing for cation exchange chromatography, as described previously (14,15). After purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high-performance liquid chromatography (SE-HPLC) under native conditions were used to determine the apparent molecular weight and purity. ...
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.
... No reuse allowed without permission. (31). In the present study, we aimed to validate whether the SL335-fused human recombinant IL-18BP could induce potent therapeutic IL-18 blockade by undertaking pharmacokinetic (PK) studies in healthy mice, rats, and cynomolgus monkeys and pharmacodynamics (PD) studies in both MAS and AD model mice. ...
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. 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.
Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation.
Key points
• The recombinant therapeutic protein degradation in CHO cell systems is reviewed.
• Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation.
• Reducing the degradation can improve the quality of recombinant therapeutic proteins.
Human serum albumin (HSA) is the most abundant protein in the blood and has desirable properties as a drug carrier. One of the most promising ways to exploit HSA as a carrier is to append an albumin-binding moiety (ABM) to a drug for in situ HSA binding upon administration. Nature- and library-derived ABMs vary in size, affinity, and epitope, differentially improving the pharmacokinetics of an appended drug. In this review, we evaluate the current state of knowledge regarding various aspects of ABMs and the unique advantages of ABM-mediated drug delivery. Furthermore, we discuss how ABMs can be specifically modulated to maximize potential benefits in clinical development. Teaser Unleashing the potential of albumin-binding moieties (ABMs): Discover the molecular diversity and therapeutic power of ABMs in prolonging drug half-life. A groundbreaking review exploring the hidden gems of ABMs, revolutionizing long-acting therapeutics.
Interferons (IFN) are immunomodulating, antiviral and antiproliferative cytokines for treatment of multiple indications, including cancer, hepatitis, and autoimmune disease. The first IFNs were discovered in 1957, first approved in 1986, and are nowadays listed in the WHO model list of essential Medicines. Three classes of IFNs are known; IFN–α2a and IFN-β belonging to type-I IFNs, IFN-γ a type-II IFN approved for some hereditary diseases and IFN-λs, which form the newest class of type-III IFNs. IFN-λs were discovered in the last decade with fascinating yet under discovered pharmaceutical potential. This article reviews available IFN drugs, their field and route of application, while also outlining available and future strategies for bioconjugation to further optimize pharmaceutical and clinical performances of all three available IFN classes.
Off-label use of a human granulocyte colony stimulating factor (hG-CSF) has been allowed to treat dogs and cats with neutropenia. However, repeated administration of hG-CSF induces undesirable anti-drug antibody (ADA) responses, implying the necessity of animal-derived G-CSF as a therapeutic reagent, preferably with a long-acting capability. Herein, we generated a recombinant fusion protein by genetically combining FL335, a chimeric Fab specific for feline serum albumin (FSA), and feline G-CSF (fG-CSF), with the ultimate goal of developing a long-acting therapeutic fG-CSF for cats. The resulting FL335-fG-CSF fusion protein, referred to as APB-F1, was produced well as a functional form in a Chinese hamster ovary (CHO) expression system. In in vitro analyses, APB-F1 bound to FSA at high affinity (KD = 400 pM) and possessed 0.78 × 10⁷ U/mg G-CSF biological activity, clearly proving its biological functionality. Pharmacokinetic (PK) and pharmacodynamic (PD) studies using healthy cats revealed that the serum half-life (t1/2) of APB-F1 was increased five times compared with that of fG-CSF (t1/2 = 13.3 h vs. 2.7 h) in subcutaneous (SC) injections. Additionally, APB-F1 induced a profound and sustained increase in white blood cell (WBC) and actual neutrophil count (ANC) up to 10 days, which was far superior to other G-CSF preparations, including filgrastim (NeupogenTM) and even pegfilgrastim (NeulastaTM). Conclusively, a long-acting fG-CSF with potent in vivo bioactivity was successfully created by using FL335; thus, we provided evidence that our “anti-serum albumin Fab-associated” (SAFA) technology can be applied reliably in developing valuable long-acting biologics in veterinary medicine.
Human serum albumin (HSA) has been used to extend the serum half-lives of various protein therapeutics through genetic fusion because HSA exhibits an exceptionally long circulation time as a result of neonatal Fc receptor (FcRn)-mediated recycling. As another serum half-life extender, the human antibody Fab SL335 that strongly binds HSA was developed. When SL335 was fused to a protein therapeutic, SL335 was shown to prolong the half-life of the drug. Despite the significance of SL335-HSA binding in the extension of drug circulation time, it remains unclear how SL335 interacts with HSA at a molecular structural level. To reveal the structural basis of HSA recognition by SL335, we determined the crystal structure of the SL335-HSA complex at a resolution of 2.95 Å. SL335 binds HSA at a 1:1 stoichiometry. SL335 uses the exposed loops of its heavy and light chains to specifically recognize the IIa and IIb subdomains of HSA. The SL335 epitope is located on the opposite side of the FcRn-binding site and does not overlap with it, suggesting that SL335 extends the serum half-lives of itself and its fusion partner through an FcRn-dependent recycling mechanism.
Interferon (IFN) beta-1b was the first disease-modifying therapy to be approved for the treatment of multiple sclerosis (MS), and over 21 years of follow-up data demonstrate its efficacy and long-term safety profile. Following recent regulatory approvals in the USA and European Union, IFN beta-1b is now one of the seven disease-modifying therapies [intramuscular IFN beta-1a; subcutaneous (SC) IFN beta-1a; IFN beta-1b SC; glatiramer acetate SC; oral dimethyl fumarate; oral teriflunomide; and intravenous alemtuzumab] indicated for first-line use in relapsing–remitting MS. Here we review the clinical trial and follow-up data for IFN beta-1b and discuss factors that clinicians may consider when selecting this treatment, both at first line in early MS, and later in the disease course.
Electronic supplementary material
The online version of this article (doi:10.1007/s12325-014-0149-1) contains supplementary material, which is available to authorized users.
Current standard immunomodulatory therapy with interferons (IFNs) for relapsing-remitting multiple sclerosis (MS) exhibits proven, but limited, efficacy and increased side effects due to the need of frequent application of the drug. Therefore, there is a need for more effective and tolerable drugs. Due to their small size, optimization of therapy with IFNs in MS by PEGylation is feasible. PEGylation of an IFN means that at least one molecule of polyethylene glycol (PEG) is covalently added. This modification is a standard procedure to increase the stability, solubility, half-life, and efficacy of a drug, and is applied in several drugs and diseases. Currently, a therapy regimen applying PEG-IFN beta-1a in MS is being developed to achieve an optimized relationship between therapy-related side effects and pharmacokinetic/pharmacodynamic efficacy. Phase I studies demonstrated that subcutaneous PEG-IFN beta-1a at a dose of 125 μg every 2 or 4 weeks might be at least as efficient and safe as the current standard therapy with IFN beta-1a. A global Phase III clinical study is investigating the efficacy of PEG-IFN beta-1a in terms of reduction of the relapse rate in relapsing-remitting MS patients. The latest primary safety and efficacy analysis after 1 year has revealed a favorable risk-benefit profile with no significant difference between dosing regimens. Compared to placebo, the annualized relapse rate was reduced by about one-third and new or newly enlarging T2 brain lesions were reduced by about one-third when dosing every 4 weeks or by two-thirds when dosing every 2 weeks. This presents a significant effect of the dosing interval, favoring administration every 2 weeks. Chronic administration of PEGylated proteins mostly at toxic concentrations causes vacuolation of renal epithelium in animals, which - along with the issue of occurrence of anti-PEG antibodies - has to be addressed by Phase IV studies.
To study the influence of protein aggregation on the immunogenicity of recombinant human interferon beta (rhIFNbeta) in wild-type mice and transgenic, immune-tolerant mice, and to evaluate the induction of immunological memory.
RhIFNbeta-1b and three rhIFNbeta-1a preparations with different aggregate levels were injected intraperitoneally in mice 15x during 3 weeks, and the mice were rechallenged with rhIFNbeta-1a. The formation of binding (BABs) and neutralizing antibodies (NABs) was monitored.
Bulk rhIFNbeta-1a contained large, mainly non-covalent aggregates and stressed rhIFNbeta-1a mainly covalent, homogeneous (ca. 100 nm) aggregates. Reformulated rhIFNbeta-1a was essentially aggregate-free. All products induced BABs and NABs in wild-type mice. Immunogenicity in the transgenic mice was product dependent. RhIFNbeta-1b showed the highest and reformulated rhIFNbeta-1a the lowest immunogenicity. In contrast with wild-type mice, transgenic mice did not show NABs, nor did they respond to the rechallenge.
The immunogenicity of the products in transgenic mice, unlike in wild-type mice, varied. In the transgenic mice, neither NABs nor immunological memory developed. The immunogenicity of rhIFNbeta in a model reflecting the human immune system depends on the presence and the characteristics of aggregates.
Macroporous microcarriers such as Cytopore entrap mammalian cells in a mesh network allowing growth to high cell concentrations in a protected environment within a stirred culture. Chinese hamster ovary (CHO) cells producing recombinant human beta-interferon (IFN-beta) and grown in Cytopore microcarriers showed a 2.6- to 2.8-fold increase in the volumetric product titer compared with cells grown in an equivalent suspension culture. In an attempt to maximize production of IFN-beta, microcarrier cultures were subjected to a low temperature regime. Low temperature culture conditions (32 degrees C) have been shown previously to enhance cell specific productivity in suspension cultures although at reduced cell growth rates. These conditions can be optimized by a timely shift from physiological to hypothermic conditions during the culture run to maximize volumetric protein production. In the case of IFN-beta production the lower temperature has the added advantage of stabilizing the product and reducing intramolecular aggregation. Using a biphasic temperature-shift regime from 37 to 32 degrees C the volumetric production of IFN-beta was enhanced to 4.2-fold compared with a single temperature suspension culture in a controlled bench-top bioreactor. Furthermore, the degree of intramolecular aggregation of IFN-beta was reduced significantly (59%) compared with control cultures, largely due to the lower temperature but also partially due to the presence of microcarriers. These results indicate that the hypothermic conditions in a Cytopore culture had a combined and possibly synergistic effect of increasing volumetric production of the recombinant protein.
The asparagine-linked sugar chains of natural interferon-beta 1 secreted from human foreskin fibroblasts by poly I:poly C induction and of three recombinant human interferon-beta 1 produced by Chinese hamster ovary cells, mouse epithelial cells (C127), and human lung adenocarcinoma cells (PC8) were released quantitatively as oligosaccharides by hydrazinolysis followed by N-acetylation. After being reduced with either NaB3H4 or NaB2H4, their structures were comparatively analyzed. More than 80% of the sugar chains of natural interferon-beta 1 occur as biantennary complex-type sugar chains, approximately 10% of which contain N-acetyllactosamine repeating structure in their outer chain moieties. The remainders are 2,4- and 2,6-branched triantennary complex-type sugar chains. The sugar chains of the recombinant interferon-beta 1 derived from Chinese hamster ovary cells were very similar to those of its natural counterpart. In contrast, two other recombinant proteins contain quite different sugar chains. The protein derived from C127 cells contains complex-type sugar chains with the Gal alpha 1----3Gal beta 1----4GlcNAc group in their outer chain moieties. Their sialic acid residues occur solely as the Sia alpha 2----6Gal group, where Sia is sialic acid. In contrast, the sialic acid residues of other interferon-beta 1 occur as the Sia alpha 2----3Gal group only. A part of the sugar chains of the protein derived from PC8 cells contains bisecting N-acetylglucosamine residue in addition to the Gal alpha 1----3Gal beta 1----4GlcNAc group.
The human fibroblast interferon gene was inserted in a thermoinducible expression plasmid under control of the phage lambda PL promoter. The primary translation products predicted on the basis of the plasmid constructions were hybrid proteins starting with beta-lactamase or phage MS2 polymerase information followed by the total preinterferon. On induction, antiviral activity, whose physico-chemical, immunological and biological characteristics closely corresponded to those of authentic human fibroblast interferon, was synthesized. Processing to a size compatible with mature but unglycosylated authentic product was observed.
Two recombinant IFN-beta products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN-beta-1a) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-beta-1b). The structural basis for activity differences between IFN-beta-1a and IFN-beta-1b, is determined.
In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN-beta products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences.
IFN-beta-1a was 10 times more active than IFN-beta-1b with specific activities in a standard antiviral assay of 20 x 10(7) IU/mg for IFN-beta-1a and 2 x 10(7) IU/mg for IFN-beta-1b. Of the known structural differences between IFN-beta-1a and IFN-beta-1b, only glycosylation affected in vitro activity. Deglycosylation of IFN-beta-1a produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN-beta-1b revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 x 10(7) IU/mg). Crystallographic data for IFN-beta-1a revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure.
Together these results suggest that the greater biological activity of IFN-beta-1a is due to a stabilizing effect of the carbohydrate on structure.
The enhancement of recombinant protein expression of a transfected cell line is essential for the development of an efficient large-scale bioprocess. The effect of various media additives and temperature conditions were studied in an attempt to optimize protein production, stability, and protein glycosylation from a Chinese hamster ovary (CHO) cell line producing human beta-interferon (Hu-beta-IFN). We observed a decrease in the ELISA response of the glycoprotein in the later stages of batch cultures, which was attributed to molecular aggregation. Cells were subjected to various concentrations of glycerol, dimethyl sulfoxide (DMSO), and sodium butyrate (NaBu) in a variety of culture systems and conditions. The addition of both NaBu and DMSO resulted in higher specific productivities but reduced growth rates that resulted in a net reduction of interferon produced. Glycerol appeared to stabilize the secreted beta-IFN, resulting in reduced aggregation, despite a decrease in cell growth rate. Glycosylation analysis of isolated beta-IFN showed a time-dependent decrease in sialylation in batch culture that was ameliorated by the presence of glycerol. Low-temperature conditions (30 degrees C) had the greatest effect on productivity with a significant increase in beta-IFN titer as well as a reduction in the degree of molecular aggregation.
Recombinant human interferon-beta (rhIFN-beta) is the leading therapeutic intervention shown to change the cause of relapsing-remitting multiple sclerosis, and both a nonglycosylated and a significantly more active glycosylated variant of rhIFN-beta are used in treatment. This study investigates the function of the rhIFN-beta1a glycan moiety and its individual carbohydrate residues, using the myxovirus resistance (Mx) mRNA as a biomarker in Mx-congenic mice. We showed that the Mx mRNA level in blood leukocytes peaked 3 h after s.c. administration of rhIFN-beta1a. In addition, a clear dose-response relationship was confirmed, and the Mx response was shown to be receptor-mediated. Using specific glycosidases, different glycosylation analogs of rhIFN-beta1a were obtained, and their activities were determined. The glycosylated rhIFN-beta1a showed significantly higher activity than its deglycosylated counterpart, due to a protein stabilization/solubilization effect of the glycan. It is interesting to note that the terminating sialic acids were essential for these effects. Conclusively, the structure/bioactivity relationship of rhIFN-beta1a was determined in vivo, and it provided a novel insight into the role of the rhIFN-beta1a glycan and its carbohydrate residues. The possibilities of improving the pharmacological properties of rhIFN-beta1a using glycoengineering are discussed.
Introduction:
Many of the biotherapeutics approved or under development suffer from a short half-life necessitating frequent applications in order to maintain a therapeutic concentration over an extended period of time. The implementation of half-life extension strategies allows the generation of long-lasting therapeutics with improved pharmacokinetic and pharmacodynamic properties. Areas covered: This review gives an overview of the different half-life extension strategies developed over the past years and their application to generate next-generation biotherapeutics. It focuses on srategies already used in approved drugs and drugs that are in clinical development. These strategies include those aimed at increasing the hydrodynamic radius of the biotherapeutic and strategies which further implement recycling by the neonatal Fc receptor (FcRn). Expert opinion: Half-life extension strategies have become an integral part of development for many biotherapeutics. A diverse set of these strategies is available for the fine-tuning of half-life and adaption to the intended treatment modality and disease. Currently, half-life extension is dominated by strategies utilizing albumin binding or fusion, fusion to an immunoglobulin Fcγ region and PEGylation. However, a variety of alternative strategies, such as fusion of flexible polypeptide chains as PEG mimetic substitute, have reached advanced stages and offer further alternatives for half-life extension.
Modification of biopharmaceutical molecules by covalent conjugation of polyethylene glycol (PEG) molecules is known to enhance pharmacologic and pharmaceutical properties of proteins and other large molecules and has been used successfully in 12 approved drugs. Both linear and branched-chain PEG reagents with molecular sizes of up to 40 kDa have been used with a variety of different PEG derivatives with different linker chemistries. This review describes the properties of PEG itself, the history and evolution of PEGylation chemistry, and provides examples of PEGylated drugs with an established medical history. A trend toward the use of complex PEG architectures and larger PEG polymers, but with very pure and well-characterized PEG reagents is described. Nonclinical toxicology findings related to PEG in approved PEGylated biopharmaceuticals are summarized. The effect attributed to the PEG part of the molecules as observed in 5 of the 12 marketed products was cellular vacuolation seen microscopically mainly in phagocytic cells which is likely related to their biological function to absorb and remove particles and macromolecules from blood and tissues. Experience with marketed PEGylated products indicates that adverse effects in toxicology studies are usually related to the active part of the drug but not to the PEG moiety.
The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0→∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.
Human serum albumin (HSA) regulates the transport and availability of numerous chemical compounds and molecules in the blood vascular system. While previous HSA research has found that HSA interacts with specific varieties of ligands, new research efforts aim to expand HSA's ability to interact with more different drugs in order to improve the delivery of various pharmacological drugs. This review will cover fatty acid chain and post-translational modifications of HSA that potentially modulate how HSA interacts with various pharmacological drugs, including glycation, cysteinylation, S-nitrosylation, S-transnitrosation and S-guanylation.
Several inteferons (IFNs) have recently been introduced for the treatment of multiple sclerosis (MS). These are IFNβ-1b (Betaseron®) and 2 IFNβ-1a preparations (Avonex® and Rebif®). All 3 drugs reduce the number of acute exacerbations in relapsing remitting MS by about one-third but, because of differences in the trials and trial design, direct comparison of efficacy is difficult. Only the IFNβ-1a preparations have been demonstrated to have an effect in slowing disability. Possible reasons for disparate results with the 3 IFNβs include substantial differences in trial design, antigenicity, route of administration and bioavailability.
Adverse effects differ among the various preparations. Avonex®, which is given intramuscularly once weekly, has the fewest adverse effects and the lowest incidence of neutralising antibody formation at 5 to 7%. IFNβ-1b has the highest incidence at 30 to 39%, and Rebif® is intermediate at 13 to 24%. Additionally, IFNβ-1b and Rebif® often cause significant skin reactions, and the former causes occasional skin necrosis at injection sites, a problem not seen with Avonex®.
On the basis of the available information, the IFNβ-1a preparations are preferred in terms of tolerability, effects on exacerbation rate and disability, less frequent antibody formation and convenience. The available preparations, Avonex® and Rebif®, have shown similar effectiveness although dose, route of administration and dosage regimens differ. The administered dose of IFNβ-1b and Rebif® appears to be somewhat higher than that of Avonex®, but the differences in effect are small. Further information regarding the effect of different doses and dosage regimens with the various preparations is expected to become available over the next few years.
Interferon-β (IFN-β) products have been used for many years in the treatment of multiple sclerosis and include recombinant IFN-β-1b (Betaseron®) and IFN-β-1a (Avonex® and Rebif®). All three products lead to the formation of neutralizing antibodies (NAbs) and resulting loss of efficacy in patients but to different extents. Across several clinical trials, the reported rates of neutralizing-antibody formation were 22%-47% (Betaseron®), 5%-35% (Rebif®), and 2%-13% (Avonex®). In the current study, all products were purchased from the pharmacy and aggregates were characterized and/or quantified using size-exclusion chromatography (SEC), analytical ultracentrifugation, gel electrophoresis, and dot-blotting immunoassays. Particle characterization and counting were performed using microflow imaging, particle tracking analysis, and resonant mass measurement. Betaseron® and Rebif®, which are formulated with human serum albumin, had the greatest amount of aggregated protein and particles (e.g., 9%-15% high molecular weight species by SEC and >100,000 particles/mL by flow imaging). Avonex® was found to have the least amount of aggregated protein, with >95% monomer content by both SEC and analytical ultracentrifugation, and the particles detected in Avonex® were determined to be primarily silicone oil droplets. These results strongly suggest that protein aggregate and particle contents are key product quality attributes in a given product's propensity to elicit the production of NAbs in patients. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
ABSTRACT Multiple sclerosis (MS) is a chronic autoimmune disease of the CNS characterized by inflammation, demyelination, and axonal injury. These pathologic effects are manifested in clinical symptoms of relapse and disability. Various disease-modifying therapies have been developed in recent years to modulate the body's immune response. Among the most widely used are the beta interferons (IFNbeta). All produce comparable biological effects and are approved for the treatment of relapsing-remitting MS (RRMS). Although the precise mechanisms through which IFNbeta achieves its antiinflammatory and immunomodulatory effects remain uncertain, several modes of action have been proposed, including inhibition of T-cell activation and proliferation; apoptosis of autoreactive T cells; induction of regulatory T cells; inhibition of leukocyte migration across the blood-brain barrier; cytokine modulation; and potential antiviral activity. Endogenously produced IFNbeta in the injured brain is also now believed to contribute to mediation of antiinflammatory and regenerative effects. All these mechanisms are believed to underlie the therapeutic effect of IFNbeta in the treatment of RRMS.
The interferon (IFN) system in man regulates viral replication, cell multiplication and immune functions. Its action against viruses takes place in two stages. The first is the production of IFN by cells following stimulation by a variety of IFN inducers including viruses, and the second is the action of this IFN on other cells inducing in them an antiviral state which prevents replication of infecting viruses. A series of assays is described which evaluates these different parameters of the antiviral IFN system. An attempt was made to correlate between IFN production and cell response to IFN on the one hand, and clinical status on the other. Results show that healthy persons have little or no IFN in the blood (mean less than 4 units/ml), and that in 94% their PBMC are not in an antiviral state. On the other hand, patients with acute viral diseases have significantly increased levels of IFN in their blood (mean 150 +/- 284 units/ml), and in 70% their cells are in an antiviral state. In some seriously ill patients with viral disease, the IFN system was found to be functionally deficient and treatment with human leucocyte IFN rapidly changed this. It is concluded that the examination of several biological parameters of the IFN system including the intracellular antiviral state induced by IFN is necessary in order to better evaluate this antiviral system. This will enable the clinician to obtain optimal pharmacokinetic information for determining which cases are most likely to respond to IFN therapy, and help to monitor the efficacy of this treatment.
Multiple sclerosis (MS) is characterized by autoimmune inflammation and subsequent neurodegeneration. It is believed that early in the disease course, proinflammatory T cells that are activated in the periphery by antigen presentation cross the blood-brain barrier (BBB) into the CNS directed by various chemotaxic agents. However, to date, there has been no formal demonstration of a specific precipitating antigen. Once inside the CNS, activated T cells including T helper-1 (Th1), Th17, γδ and CD8+ types are believed to secrete proinflammatory cytokines. Decreased levels of Th2 cells also correlate with relapses and disease progression in MS, since Th2-derived cytokines are predominantly anti-inflammatory. In healthy tissue, inflammatory effects are opposed by specific subsets of regulatory T cells (Tregs) including CD4+, CD25+ and FoxP3+ cells that have the ability to downregulate the activity of proinflammatory T cells, allowing repair and recovery to generally follow inflammatory insult. Given their function, the pathogenesis of MS most likely involves deficits of Treg function, which allow autoimmune inflammation and resultant neurodegeneration to proceed relatively unchecked.
Interferons (IFNs) are naturally occurring cytokines possessing a wide range of anti-inflammatory properties. Recombinant forms of IFNβ are widely used as first-line treatment in relapsing forms of MS. The mechanism of action of IFNb is complex, involving effects at multiple levels of cellular function. IFNβ appears to directly increase expression and concentration of anti-inflammatory agents while downregulating the expression of proinflammatory cytokines. IFNβ treatment may reduce the trafficking of inflammatory cells across the BBB and increase nerve growth factor production, leading to a potential increase in neuronal survival and repair. IFNβ can also increase the number of CD56bright natural killer cells in the peripheral blood. These cells are efficient producers of anti-inflammatory mediators, and may have the ability to curb neuron inflammation. The mechanistic effects of IFNβ manifest clinically as reduced MRI lesion activity, reduced brain atrophy, increased time to reach clinically definite MS after the onset of neurological symptoms, decreased relapse rate and reduced risk of sustained disability progression.
The mechanism of action of IFNβ in MS is multifactorial and incompletely understood. Ongoing and future studies will increase our understanding of the actions of IFNβ on the immune system and the CNS, which will in turn aid advances in the management of MS.
Like many other therapeutic proteins, recombinant human interferon beta (rhIFN-β) elicits undesirable immune responses. rhIFN-β-treated multiple sclerosis patients may form binding antibodies and neutralizing antibodies (NAbs), with the latter being responsible for inhibition of the therapeutic effect of the protein. The incidence of binding antibodies and NAbs against rhIFN-β as well as the titer and persistence of NAbs differ among the marketed products. The proportion of patients forming antibodies against rhIFN-β-1b is higher than that against rhIFN-β-1a, which is likely explained by the differences in protein structure and aggregation behavior between the 2 types of rhIFN-β. Here, we summarize the different factors influencing the immunogenicity of rhIFN-β in patients with multiple sclerosis and discuss the role played by rhIFN-β aggregates.
For efficient production of native interferon-beta (IFN-beta) in recombinant CHO cell culture, the IFN-beta molecular aggregation that occurs during culture needs to be minimized. To do so, we investigated the effect of hyperosmolality and hypothermia on IFN-beta production and molecular aggregation in rCHO cell culture. Both hyperosmolality (470 mOsm/kg) and hypothermia (32 degrees C) increased specific native INF-beta productivity q(IFN-beta). Furthermore, they decreased the IFN-beta molecular aggregation, although severe IFN-beta molecular aggregation could not be avoided in the later phase of culture. To overcome growth suppression at hyperosmolality and hypothermia, cells were cultivated in a biphasic mode. Cells were first cultivated at 310 mOsm/kg and 37 degrees C for 2 days to rapidly obtain a reasonably high cell concentration. The temperature and osmolality were then shifted to 32 degrees C and 470 mOsm/kg, respectively, to achieve high q(IFN-beta) and reduced IFN-beta molecular aggregation. Due to the enhanced q(IFN-beta) and delayed molecular aggregation, the highest native IFN-beta concentration achieved on day 6 was 18.03 +/- 0.61 mg/L, which was 5.30-fold higher than that in a control batch culture (310 mOsm/kg and 37 degrees C). Taken together, a combination of hyperosmolality and hypothermia in a biphasic culture is a useful strategy for improved native IFN-beta production from rCHO cells.
Human fibroblast interferon has three cysteine residues, located at amino acid positions 17, 31, and 141. Using the technique of site-specific mutagenesis with a synthetic oligonucleotide primer, we changed the codon for cysteine-17 to a codon for serine. The resulting interferon, IFN-beta Ser-17, retains the antiviral, natural killer cell activation, and antiproliferative activities of native fibroblast interferon. The purified IFN-beta Ser-17 protein has an antiviral specific activity of 2 X 10(8) units/mg, similar to that of purified native fibroblast interferon. In addition, the purified protein is stable to long-term storage at -70 degrees C.
We performed yearly MRI analyses on 327 of the total 372 patients in a multicenter, randomized, double-blind, placebo-controlled trial of interferon beta-1b (IFNB). Clinical results are presented in the preceding companion paper. Baseline MRI characteristics were the same in all treatment groups. Fifty-two patients at one center formed a cohort for frequent MRIs (one every 6 weeks) for analysis of disease activity. The MRI results support the clinical results in showing a significant reduction in disease activity as measured by numbers of active scans (median 80% reduction, p = 0.0082) and appearance of new lesions. In addition, there was an equally significant reduction in MRI-detected burden of disease in the treatment as compared with placebo groups (mean group difference of 23%, p = 0.001). These results demonstrate that IFNB has made a significant impact on the natural history of MS in these patients.
Interferon therapies suffer from a relatively short half-life of the products in circulation. To address this issue we investigated the effects of polyethylene glycol modification (PEGylation) on the pharmacokinetic properties of human interferon (IFN)-beta-1a. PEGylation with a linear 20-kDa PEG targeted at a single site on the N-terminal amine had no deleterious effect on its specific activity in an in vitro antiviral assay. In monkeys, PEG IFN-beta-1a treatment induced neopterin and beta2-microglobulin expression (pharmacodynamic markers of activity). Systemic clearance values in monkeys, rats, and mice decreased, respectively, from 232, 261, and 247 ml/h/kg for the unmodified IFN-beta-1a to 30.5, 19.2, and 18.7 ml/h/kg for the PEGylated form, while volume of distribution values decreased from 427, 280, and 328 ml/kg to 284, 173, and 150 ml/kg. The decreased clearance and volume of distribution resulted in higher serum antiviral activity in the PEG IFN-beta-1a-treated animals. In the rat, a more extensive set of dosing routes was investigated, including intraperitoneal, intratracheal, and oral administration. Bioavailability for the PEG IFN-beta-1a was similar to the unmodified protein for each of the extravascular routes examined. For the intraperitoneal route, bioavailability was almost 100%, whereas for the oral and intratracheal routes absorption was low (<5%). In rats, subcutaneous bioavailability was moderate (28%), whereas in monkeys it was approximately 100%. In all instances an improved pharmacokinetic profile for the PEGylated IFN-beta-1a was observed. These findings demonstrate that PEGylation greatly alters the pharmacokinetic properties of IFN-beta-1a, resulting in an increase in systemic exposure following diverse routes of administration.
Recent clinical trials with interferon-beta (IFN-beta) in relapsing-remitting multiple sclerosis (RRMS) have clearly demonstrated that the IFN-beta dosing regimen affects the clinical efficacy, thereby highlighting the importance of determining the relative biologic activities of the IFN-beta products currently available. Although studies have been published that examine the biologic activities of the two structurally different forms of recombinant IFN-beta, IFN-beta1a (Rebif), Serono, Geneva, Switzerland) and IFN-beta1b (Betaseron)/Betaferon), Berlex [Montville, NJ]/Schering [Berlin, Germany]), there have been few direct comparative studies. Therefore, to obtain a more accurate estimate of the relative biologic activities of Rebif and Betaseron, this study examined the antiviral activities of these two products within the same assay system and against the same natural human IFN-beta standard. Whereas the manufacturers' information suggests that the bioactivity of Betaseron is only about 8.7-fold less than that of Rebif, the results of the present direct, comparative study show that Rebif has an antiviral activity 14 times greater than that of Betaseron. This may have important clinical implications, because on the basis of the results reported here, Rebif at 44 microg t.i.w. is approximately double the maximal licensed weekly dose for Betaseron.
The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.
It is now understood that the nonclassical major histocompatibility complex-I molecule FcRn binds albumin and retrieves it from an intracellular degradative fate. Whether FcRn in the liver modulates albumin turnover through effects on biosynthesis and production is not known. Thus we quantified the appearance of biosynthetically labeled albumin in plasma after an intravenous bolus injection of [(3)H]leucine in FcRn-deficient mice. The production rates for both albumin (FcRn substrate) and transferrin (nonsubstrate) are increased by approximately 20% in FcRn-deficient mice compared with normal mice, likely compensating for the lowered plasma oncotic pressure caused by hypoalbuminemia in FcRn-deficient mice. Determining the magnitude of FcRn-mediated effects on albumin turnover, we then measured the steady-state plasma concentrations of biosynthetically labeled albumin and transferrin during [(3)H]leucine infusion. The concentration of albumin was approximately 40% lower in FcRn-deficient mice compared with normal mice. Furthermore, the approximately 40% lower plasma albumin concentration in FcRn-deficient mice along with the approximately 20% increase in albumin production indicate, by the mass-balance equation, that albumin degradation in FcRn-deficient mice is twice that of normal mice. These studies of biosynthetically labeled, and thus native, albumin support our previous finding that FcRn protects albumin from degradation. Permitting quantification of the magnitude of FcRn-mediated recycling, they further indicate that FcRn has extraordinary capacity: the amount of albumin saved from degradation by FcRn-mediated recycling is the same as that produced by the liver.
PEGylation of IFN-alpha has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-beta-1a (IFN-beta-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal alpha-amino group of the cytokine. The PEGylated protein retained approximately 50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced approximately 30-fold and approximately 4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was approximately 13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 x 10(6) units of unmodified IFN-beta-1a resulted in a 29% reduction in vessel number, while 1 x 10(6) units of PEGylated IFN-beta-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 x 10(6) units of unmodified IFN-beta-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 x 10(6) units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-beta is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-beta-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-beta-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.
An AB.Fab (albumin-binding Fab) consists of a Fab and a phage-derived albumin-binding peptide. This molecule is capable of binding both antigen and albumin simultaneously. Using a Fab derived from Herceptin we generated a panel of AB.Fab variants with wide-ranging affinities for albumin. An assay that measured AB.Fab binding to albumin in solution was developed to most accurately reflect the binding affinity for albumin in vivo. Affinity varied depending upon the species of albumin tested. For rat and rabbit albumin, affinities ranged from 0.04 to 2.5 microM. Reduced affinity for albumin correlated with a reduced half-life and higher clearance rates in both species; the beta half-life ranged 6-fold while clearance ranged over 50-fold in rats and 20-fold in rabbits. To estimate the pharmacokinetic properties of an AB.Fab in humans, AB.Fab variants with similar affinities for rat and rabbit albumin were selected. Using their pharmacokinetic parameters and the principles of allometric scaling for albumin, we estimate an approximate beta half-life for an AB.Fab with 0.5 microM affinity for albumin of up to 4 days in humans with a clearance of 76 ml/h. These variants demonstrate the ability to modulate the clearance of a Fab fragment in vivo and help to establish guidelines for pharmacokinetic engineering of molecules through albumin binding.
Recent guidelines have recommended the use of validated assays for the measurement of neutralizing antibodies (NABs) to interferon beta (IFNbeta) in patients with multiple sclerosis (MS). In an attempt of validation, we studied the analytical performance of a bioassay based on antiviral cytopathic effect (CPE) using WISH cells and the vesicular stomatitis virus (WISH/VSV CPE).
NAB titres measured with the WISH/VSV CPE assay in 63 sera from IFNbeta-treated MS patients were compared to those obtained with the reference CPE method using A549 cells and the encephalomyocarditis virus. Binding antibodies (BABs) were measured using a capture ELISA as a screening test for NABs.
No false-negative BAB was obtained in our patients. The between-run coefficients of variation (CVs) determined with log10 titres of the NIH anti-IFNbeta (G038-501-572) yielded good results (<or=10.4%) and within-run variability was excellent (CV<or=2%). The log10 titres obtained with both CPE assays were highly correlated (r=0.969 and r=0.884 for anti-IFNbeta-1a and anti-IFNbeta-1b, respectively). The same patients were found NAB-positive with both CPE assays.
Because of its good precision, sensitivity and excellent correlation with the reference CPE method, the WISH/VSV CPE bioassay can be used in the follow-up of IFNbeta-treated MS patients.
In patients with multiple sclerosis (MS), activation of immune cells and breakdown of the blood-brain barrier (BBB) lead to demyelination and axon injury. Although repairing damage to neurons is not possible, immunomodulating therapies can reduce the inflammatory processes that lead to demyelination. Interferon-beta (IFN-beta) is a polypeptide, normally produced by fibroblasts, that has antiviral and antiproliferative effects. Binding of IFN-beta to its receptor induces a complex transcriptional response. In immune cells (the most likely target of IFN-beta's therapeutic effect in MS), IFN-beta reduces antigen presentation and T-cell proliferation, alters cytokine and matrix metalloproteinase (MMP) expression, and restores suppressor function. Therapeutic forms of IFN-beta can be produced in bacterial expression systems (IFN-beta1b) or in mammalian cells (IFN-beta1a). These forms have some differences in their amino acid sequence and posttranslational modifications, but the transcriptional response to IFN- beta1b and IFN-beta1a appears to be similar, if not indistinguishable. However, the biological response and the clinical effect do vary with changes in the dosing frequency of IFN-beta. In clinical trials, IFN-beta1a IM administered weekly elicits a transient biological response compared to IFN-beta1b administered SC every other day or IFN-beta1a (administered SC three times per week). Comparative clinical trials suggest that the differences in the biological response are clinically meaningful: more frequent IFN-beta administration produces superior clinical responses.
Interferon beta (INFbeta) may induce the expression of several proteins, including neopterin, considered a biological marker of INFbeta activity.
The aim of this study was to determine the serum neopterin concentration at the beginning of, and during, IFNbeta-1a therapy in relapsing-remitting multiple sclerosis (r-r MS) patients, and to look for a possible correlation between protein synthesis and the clinical course of the disease.
Thirteen r-r MS patients were treated with INFbeta-1a (i.m. 6 MIU/week) for 2 years. Blood samples for neopterin determinations were taken daily over a period of 1 week at the end of each 6 months of therapy, and tested for neutralizing antibodies (NABs).
Neopterin levels peaked 24-48 h post-injection and returned to baseline after 120 h. After 1 year of therapy, four patients dropped out of the study because of progression of the disease: in these subjects a significant decrement of neopterin was observed.
Neopterin baseline values were not found to decrease over the 2 years of therapy, and neopterin may be considered to be a useful marker of responsiveness to IFNbeta.
The family of interferon (IFN) proteins has now more than reached the potential envisioned by early discovering virologists: IFNs are not only antivirals with a spectrum of clinical effectiveness against both RNA and DNA viruses, but are also the prototypic biological response modifiers for oncology, and show effectiveness in suppressing manifestations of multiple sclerosis. Studies of IFNs have resulted in fundamental insights into cellular signalling mechanisms, gene transcription and innate and acquired immunity. Further elucidation of the multitude of IFN-induced genes, as well as drug development strategies targeting IFN production via the activation of the Toll-like receptors (TLRs), will almost certainly lead to newer and more efficacious therapeutics. Our goal is to offer a molecular and clinical perspective that will enable IFNs or their TLR agonist inducers to reach their full clinical potential.
We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum
albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding
dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa ‘AlbudAbs’ can match the half-life
of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of
the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb. Soluble IL-1ra is potent inhibitor of IL-1 signalling that
is approved for the treatment of rheumatoid arthritis but has a relatively short in vivo half-life. Here we show that although the AlbudAb/IL-1ra fusion has a similar in vitro potency, its in vivo efficacy can be dramatically improved due to its extended serum half-life. AlbudAbs could potentially be used to generate
a range of long half-life versions of many different drugs in order to improve their dosing regimen and/or clinical effect.