Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
2.
Brain Behav Immun Health ; 16: 100307, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34589799

ABSTRACT

The proinflammatory cytokine Interleukin-1 (IL-1), with its two isoforms α and ß, has important roles in multiple pathogenic processes in the central nervous system. The present study aimed to evaluate and compare the blood-to-brain distribution of anakinra (IL-1 receptor antagonist), bermekimab (IL-1α antagonist) and canakinumab (IL-1ß antagonist). A human in vitro model of the blood-brain barrier derived from human umbilical cord blood stem cells was used, where isolated CD34+ cells co-cultured with bovine pericytes were matured into polarized brain-like endothelial cells. Transport rates of the three test items were evaluated after 180 â€‹min incubation at concentrations 50, 250 and 1250 â€‹nM in a transwell system. We report herein that anakinra passes the human brain-like endothelial monolayer at a 4-7-fold higher rate than the monoclonal antibodies tested. Both antibodies had similar transport rates at all concentrations. No dose-dependent effects in transport rates were observed, nor any saturation effects at supraphysiological concentrations. The larger propensity of anakinra to pass this model of the human blood-brain barrier supports existing data and confirms that anakinra can reach the brain compartment at clinically relevant concentrations. As anakinra inhibits the actions of both IL-1α and IL-1ß, it blocks all effects of IL-1 downstream signaling. The results herein further add to the growing body of evidence of the potential utility of anakinra to treat neuroinflammatory disorders.

3.
Mol Pharm ; 18(1): 328-337, 2021 01 04.
Article in English | MEDLINE | ID: mdl-33259222

ABSTRACT

Albumin-binding fusion partners are frequently used as a means for the in vivo half-life extension of small therapeutic molecules that would normally be cleared very rapidly from circulation. However, in applications where small size is key, fusion to an additional molecule can be disadvantageous. Albumin-derived affinity proteins (ADAPTs) are a new type of scaffold proteins based on one of the albumin-binding domains of streptococcal protein G, with engineered binding specificities against numerous targets. Here, we engineered this scaffold further and showed that this domain, as small as 6 kDa, can harbor two distinct binding surfaces and utilize them to interact with two targets simultaneously. These novel ADAPTs were developed to possess affinity toward both serum albumin as well as another clinically relevant target, thus circumventing the need for an albumin-binding fusion partner. To accomplish this, we designed a phage display library and used it to successfully select for single-domain bispecific binders toward a panel of targets: TNFα, prostate-specific antigen (PSA), C-reactive protein (CRP), renin, angiogenin, myeloid-derived growth factor (MYDGF), and insulin. Apart from successfully identifying bispecific binders for all targets, we also demonstrated the formation of the ternary complex consisting of the ADAPT together with albumin and each of the five targets, TNFα, PSA, angiogenin, MYDGF, and insulin. This simultaneous binding of albumin and other targets presents an opportunity to combine the advantages of small molecules with those of larger ones allowing for lower cost of goods and noninvasive administration routes while still maintaining a sufficient in vivo half-life.


Subject(s)
Recombinant Fusion Proteins/metabolism , Serum Albumin/metabolism , Bacterial Proteins/metabolism , Half-Life , Life Expectancy , Protein Binding/physiology , Streptococcus/metabolism , Tumor Necrosis Factor-alpha/metabolism
4.
Methods Mol Biol ; 2178: 159-166, 2021.
Article in English | MEDLINE | ID: mdl-33128750

ABSTRACT

In this chapter, we present an efficient method for stringent protein purification facilitated by a dual affinity tag referred to as ABDz1, which is based on a 5 kDa albumin-binding domain from Streptococcal Protein G. The small fusion tag enables an orthogonal affinity purification approach based on two successive and highly specific affinity purification steps. This approach is enabled by native binding of ABDz1 to human serum albumin and engineered binding to Staphylococcal Protein A, respectively. The ABDz1-tag can be fused to either terminus of a protein of interest and the purification steps can be carried out using standard laboratory equipment.


Subject(s)
Bacterial Proteins , Recombinant Fusion Proteins , Serum Albumin, Human , Staphylococcal Protein A , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Serum Albumin, Human/chemistry , Serum Albumin, Human/genetics , Serum Albumin, Human/isolation & purification , Staphylococcal Protein A/chemistry , Staphylococcal Protein A/genetics , Staphylococcal Protein A/isolation & purification
5.
Protein Eng Des Sel ; 29(5): 187-95, 2016 May.
Article in English | MEDLINE | ID: mdl-26984961

ABSTRACT

During the past decades, advances in protein engineering have resulted in the development of variousin vitroselection techniques (e.g. phage display) to facilitate discovery of new and improved proteins. The methods are based on linkage between genotype and phenotype and are often performed in successive rounds of selection. Since the resulting output depends on the selection pressures used and the applied strategy, parameters in each round must be carefully considered. In addition, studies have reported biases that can cause enrichment of unwanted clones and/or low correlation between abundance in output and affinity. We have recently developed a selection method based on display of protein libraries onStaphylococcus carnosusand isolation of affinity proteins by fluorescence-activated cell sorting. Here, we compared duplicate selections for affinity maturation using equilibrium binding at different target concentrations and kinetic off-rate selection. The results showed that kinetic selection is efficient for isolation of high-affinity binders and that equilibrium selection at subnanomolar concentrations should be avoided. Furthermore, the reproducibility of the selection was high and a clear correlation was observed between enrichment and affinity. This work reports on the reproducibility of bacterial display in combination with FACS and provides insights into selection design to help guide the development of new affinity proteins.


Subject(s)
Cell Surface Display Techniques/methods , Flow Cytometry/methods , Peptide Library , Staphylococcus/genetics , Amino Acid Sequence , Humans , Models, Molecular , Protein Engineering , Protein Structure, Secondary , Receptor, ErbB-3/chemistry , Receptor, ErbB-3/genetics , Receptor, ErbB-3/isolation & purification , Reproducibility of Results , Sequence Analysis, DNA , Surface Plasmon Resonance
6.
Bioconjug Chem ; 27(3): 716-26, 2016 Mar 16.
Article in English | MEDLINE | ID: mdl-26781756

ABSTRACT

Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with (99m)Tc(CO)3. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)3-tag instead of the commonly used hexahistidine (H6)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H6- or (HE)3-tags in the N-termini were prepared. The constructs, (HE)3-ADAPT6 and H6-ADAPT6, were labeled with two different nuclides, (99m)Tc or (111)In. The labeling with (99m)Tc(CO)3 utilized the histidine-containing tags, while (111)In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For (111)In-labeled ADAPTs, the use of (HE)3 provided a significantly (p < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of (111)In-(HE)3-ADAPT6 and H6-ADAPT6 at later time points. Interestingly, in the case of (99m)Tc, (99m)Tc(CO)3-H6-ADAPT6 provided significantly (p < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP.


Subject(s)
Histidine/chemistry , Proteins/chemistry , Amino Acid Sequence , Tissue Distribution
7.
Cancer Res ; 75(20): 4364-71, 2015 Oct 15.
Article in English | MEDLINE | ID: mdl-26297736

ABSTRACT

Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, ¹¹¹In for SPECT imaging and 68Ga for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule (111)In/68Ga-DOTA-(HE)3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.


Subject(s)
Gene Expression , Molecular Imaging , Neoplasms/diagnostic imaging , Neoplasms/genetics , Protein Interaction Domains and Motifs/genetics , Proteins/chemistry , Proteins/genetics , Animals , Carrier Proteins/metabolism , Cell Line, Tumor , Disease Models, Animal , Female , Heterografts , Humans , Isotope Labeling , Mice , Neoplasm Metastasis , Neoplasms/metabolism , Neoplasms/pathology , Protein Binding , Proteins/metabolism , Radionuclide Imaging , Receptor, ErbB-2/metabolism , Tissue Distribution
8.
PLoS One ; 9(8): e103094, 2014.
Article in English | MEDLINE | ID: mdl-25089830

ABSTRACT

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.


Subject(s)
Albumins/metabolism , Antibodies, Bispecific/immunology , Antibody Affinity/immunology , Protein Engineering , Receptor, ErbB-2/metabolism , Amino Acid Sequence , Antibodies, Monoclonal, Humanized/metabolism , Binding, Competitive , Cell Line, Tumor , Cell Surface Display Techniques , Flow Cytometry , Humans , Molecular Sequence Data , Protein Binding , Receptor, ErbB-2/chemistry , Transition Temperature , Trastuzumab
9.
Methods Mol Biol ; 1129: 205-10, 2014.
Article in English | MEDLINE | ID: mdl-24648079

ABSTRACT

Protein fusion tags are important tools in research when robust methods for protein purification and detection are required. In this chapter we present an efficient method for stringent protein purification. A small domain, denoted ABDz1, with affinity for both human serum albumin and Protein A has been developed. The purification tag is based on an albumin-binding domain from Streptococcal Protein G that was engineered to bind Protein A. The ABDz1-tag can be fused to any protein of choice and the purification can be performed using standard laboratory equipment. In this chapter a method for purification of ABDz1-tagged proteins using two successive affinity purification steps is described.


Subject(s)
Proteins/isolation & purification , Chromatography, Affinity/methods , Protein Binding
10.
Cell Mol Life Sci ; 70(20): 3973-85, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23728098

ABSTRACT

Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal Protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.


Subject(s)
Bacterial Proteins/metabolism , Receptor, ErbB-3/metabolism , Serum Albumin/metabolism , Bacterial Proteins/genetics , Binding, Competitive , Biosensing Techniques , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Ligands , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Peptide Library , Protein Binding , Protein Interaction Domains and Motifs , Protein Stability , Protein Structure, Secondary , Receptor, ErbB-3/genetics , Serum Albumin, Human , Temperature
SELECTION OF CITATIONS
SEARCH DETAIL
...