| 1. |
- Uhlén, Mathias, et al.
(author)
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The human secretome
- 2019
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In: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 12:609
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Journal article (peer-reviewed)abstract
- The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.
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| 2. |
- Dannemeyer, Melanie, et al.
(author)
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Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors
- 2024
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 221
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Journal article (peer-reviewed)abstract
- Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply. Commonly, amounts of several hundreds of milligrams to grams are needed for different experiments, including structural investigations and activity evaluations, which require rather large cultivation volumes. This implies that cultivation of large volumes of either transiently transfected cells or stable pools/stable cell lines is needed. Hence, a production process that is scalable, speeds up the development projects, and increases the robustness of protein reagent quality throughout scales. Here we present a protein production pipeline with high scalability. We show that our protocols for protein production in Chinese hamster ovary cells allow for a seamless and efficient scale-up with robust product quality and high performance. The flexible scale of the production process, as shown here, allows for shorter lead times in drug discovery projects where there is a reagent demand for a specific protein or a set of target proteins.
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| 3. |
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| 4. |
- Jönsson, Malin, et al.
(author)
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CaRA – A multi-purpose phage display library for selection of calcium-regulated affinity proteins
- 2022
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In: New Biotechnology. - : Elsevier B.V.. - 1871-6784 .- 1876-4347. ; 72, s. 159-167
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Journal article (peer-reviewed)abstract
- Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could subsequently be of great use in gentle protein purification or as novel therapeutic modalities.
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| 5. |
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| 6. |
- Kanje, Sara, 1986-, et al.
(author)
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Engineering of Protein A for improved purification of antibodies and Fc-fused proteins
- 2020
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In: Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics. - : Elsevier. ; , s. 35-54
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Book chapter (other academic/artistic)abstract
- In industrial scale downstream processing of antibodies and Fc-fusion proteins, Protein A chromatography is the most commonly used purification method. As the treatments for many severe diseases shift toward biological drugs, in particular antibodies, the need for robust purification methods has increased. Since the 1970s, when Protein A debuted as an affinity ligand, the full protein and its domains have been extensively studied. Even if matrices based on unmodified Protein A historically met the process demands of industrial antibody manufacturing, there is room for improvement. The cleaning in place (CIP) methods required for safe reuse of the matrix in therapeutic protein purification pose a problem since the harsh conditions required can be harmful to the proteinaceous purification ligand. Further, the low pH used for elution can sometimes be detrimental to the target protein. Here, efforts to improve these features as well as capacity improvements for Protein A resins will be discussed.
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| 7. |
- Kanje, Sara, 1986-
(author)
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Engineering of small IgG binding domains for antibody labelling and purification
- 2016
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Doctoral thesis (other academic/artistic)abstract
- In protein engineering, rational design and selection from combinatorial libraries are methods used to develop proteins with new or improved features. A very important protein for the biological sciences is the antibody that is used as a detecting agent in numerous laboratory assays. Antibodies used for these purposes are often ”man-made”, by immunising animals with the desired target, or by selections from combinatorial libraries. Naturally, antibodies are part of the immune defence protecting us from foreign attacks from e.g. bacteria or viruses. Some bacteria have evolved surface proteins that can bind to proteins abundant in the blood, like antibodies and serum albumin. By doing so, the bacteria can cover themselves in the host’s own proteins and through that evade being detected by the immune system. Two such proteins are Protein A from Staphylococcus aureus and Protein G from group C and G Streptococci. Both these proteins contain domains that bind to antibodies, one of which is denoted C2 (from Protein G) and another B (from Protein A). The B domain have been further engineered to the Z domain.In this thesis protein engineering has been used to develop variants of the C2 and Z domains for site-specific labelling of antibodies and for antibody purification with mild elution. By taking advantage of the domains’ inherent affinity for antibodies, engineering and design of certain amino acids or protein motifs of the domains have resulted in proteins with new properties. A photo crosslinking amino acid, p-benzoylphenylalanine, have been introduced at different positions to the C2 domain, rendering three new protein domains that can be used for site-specific labelling of antibodies at the Fc or Fab fragment. These domains were used for labelling antibodies with lanthanides and used for detection in a multiplex immunoassay. Moreover, a library of calcium-binding loops was grafted onto the Z domain and used for selection of a domain that binds antibodies in a calcium dependent manner. This engineered protein domain can be used for the purification of antibodies using milder elution conditions, by calcium removal, as compared to traditional antibody purification.
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| 8. |
- Kanje, Sara, 1986-, et al.
(author)
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Improvements of a high-throughput protein purification process using a calcium-dependent setup
- 2020
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 175
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Journal article (peer-reviewed)abstract
- The Human Secretome Project aims to produce and purify all human secreted proteins as full-length. In order to enable this, a robust, gentle and effective purification process is needed, where multiple proteins can be purified in parallel. For this reason, a purification system based on a Protein C-tag and the HPC4 antibody with high affinity to the tag was chosen for purification. The strong binding between the tag and the antibody is specific and calcium-dependent, which allows for mild elution with EDTA. Presented here is a study comparing different protein purification base matrices coupled with the HPC4 antibody, aiming to increase the yield of purified protein and reduce the time for purification. Among the different tested matrices, Capto XP showed a high coupling degree and increased the amount of eluted protein as compared to the control matrix. By moving from batch incubation to direct sample loading and by performing the purification on the aKTAxpress, an automated protein purification process and a high reduction of hands-on sample handling was achieved. This new method also integrates the desalting step in the purification process, and the time for purification and analysis of each sample was decreased from five to three days. Moreover, a new mild method for matrix regeneration was developed using 50 mM EDTA pH 7.5 instead of 0.1 M glycine pH 2. This method was proven to be efficient for regeneration while maintaining the column binding performance even after nine rounds of regeneration.
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| 9. |
- Kanje, Sara, 1986-, et al.
(author)
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Next generation of labeling reagents for quantitative and multiplexing immunoassays by use of LA-ICP-MS
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Other publication (other academic/artistic)abstract
- Abstract. Immuno imaging by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a growing research field in the life sciences as biology and biomedicine. Various element labeling strategies for antibodies have been developed for the application of multiplex immunoassays analyzed by use of LA-ICP-MS. High multiplexing capabilities, a wide linear dynamic range and the possibility of absolute quantification are the main advantages of ICP-MS. But in the context of immuno imaging by use of LA-ICP-MS quantification of analytes is limited due to a non-controllable antibody labeling chemistry. in the presented proof-of-principle A novel antibody labeling technique has been investigated which results in a controlled labeling degree. A small affinity protein based on the C2 domain of protein G was modified with conventional metal coded tags (MeCAT) after introducing a cysteine in the C-terminus of the protein. The modified C2 domain photo crosslinks to the Fc or Fab region of the IgG and allows specific and covalent labeling of antibodies for multiplex immunoassays analyzed by use of LA-ICP-MS. In combination with a house-made calibration membrane the amount of labeled antibody-antigen complexes in a multiplex Western Blot immunoassay was determined by LA-ICP-MS.
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| 10. |
- Kanje, Sara, 1986-, et al.
(author)
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Protein engineering allows for mild affinity-based elution of therapeutic antibodies
- 2018
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In: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 430:18, s. 3427-3438
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Journal article (peer-reviewed)abstract
- Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification
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| 11. |
- Kanje, Sara, 1986-, et al.
(author)
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Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain
- 2016
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In: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812.
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Journal article (peer-reviewed)abstract
- Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody’s binding to its target. Moreover, an emerging field for therapeutics is antibody–drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.
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| 12. |
- Nilvebrant, Johan, 1982-, et al.
(author)
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Ligand and use thereof
- 2023
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Patent (pop. science, debate, etc.)abstract
- The present invention is within the field of protein engineering and purification. The invention relates to a target-binding polypeptide mutant of an IgG binding polypeptide, such as Protein A, Protein G, Protein L or Protein M, comprising a metal binding motif. More closely the invention relates to an Fc binding ligand comprising an engineered protein based on the Protein A derived Z domain, to which a calcium binding EF-loop has been introduced.
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| 13. |
- Scheffel, Julia, et al.
(author)
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Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification
- 2019
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In: mAbs. - : TAYLOR & FRANCIS INC. - 1942-0862 .- 1942-0870.
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Journal article (peer-reviewed)abstract
- As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, Z(Ca), displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of Z(Ca) to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, Z(Ca)TetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with Z(Ca)TetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.
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| 14. |
- Scheffel, Julia, et al.
(author)
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ZCa : A protein A-derived domain with calcium-dependent affinity for mild antibody purification
- 2021. - Volume 2178
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In: Methods in Molecular Biology. - New York, NY : Humana Press Inc.. ; , s. 245-249
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Book chapter (peer-reviewed)abstract
- Therapeutic antibodies are at the forefront of modern medicine where high purity, which is typically obtained by Protein A-based affinity purification, is of utmost importance. In this chapter, we present a method for neutral and selective purification of antibodies by utilizing an engineered affinity ligand, ZCa, derived from Protein A. This domain displays a calcium-dependent binding of antibodies and has been multimerized and immobilized to a chromatography resin to achieve an affinity matrix with high binding capacity. IgG antibodies can be eluted from the tetrameric ZCa ligand at pH 7 with the addition of EDTA, or at pH 5.5 with EDTA for purification of monoclonal IgG1, which is significantly milder than the low pH (3–4) required in conventional Protein A affinity chromatography. Here, a protocol for selective capture of IgG with elution at neutral pH from a ZCa tetramer ligand immobilized on a chromatography resin is described.
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| 15. |
- Tegel, Hanna, et al.
(author)
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High throughput generation of a resource of the human secretome in mammalian cells
- 2020
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In: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 58, s. 45-54
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Journal article (peer-reviewed)abstract
- The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic beta-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.
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| 16. |
- von Witting, Emma, et al.
(author)
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ADAPT as a single-domain bispecific scaffold capable of albumin-associated haf-life extension for therapeutic applications
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Other publication (other academic/artistic)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. ABD-Derived Affinity ProTeins (ADAPTs) are a type of scaffold proteins based on one of the albumin binding domains of streptococcal Protein G, with newly introduced 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 bind to 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 novel phage display library and used it to successfully select for single-domain bispecific binders towards a panel of targets: TNFa, PSA (Prostate Specific Antigen), CRP (C-reactive protein), renin, angiogenin and insulin. Apart from successfully identifying bispecific binders for all targets, we also demonstrated the formation of the ternary complex of the ADAPT together with albumin and each of the four targets TNFa, PSA, angiogenin 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 non-invasive administration routes while still maintaining a sufficient in vivo half-life.
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| 17. |
- von Witting, Emma, et al.
(author)
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Affinity-Based Methods for Site-Specific Conjugation of Antibodies
- 2021
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In: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 32, s. 1515-1524
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Research review (peer-reviewed)abstract
- Conjugation of various reagents to antibodies has long been an elegant way to combine the superior binding features of the antibody with other desired but non-natural functions. Applications range from labels for detection in different analytical assays to the creation of new drugs by conjugation to molecules which improves the pharmaceutical effect. In many of these applications, it has been proven advantageous to control both the site and the stoichiometry of the conjugation to achieve a homogeneous product with predictable, and often also improved, characteristics. For this purpose, many research groups have, during the latest decade, reported novel methods and techniques, based on small molecules, peptides, and proteins with inherent affinity for the antibody, for site-specific conjugation of antibodies. This review provides a comprehensive overview of these methods and their applications and also describes a historical perspective of the field.
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| 18. |
- von Witting, Emma, et al.
(author)
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Small Bispecific Affinity Proteins for Simultaneous Target Binding and Albumin-Associated Half-Life Extension
- 2021
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In: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 18:1, s. 328-337
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Journal article (peer-reviewed)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.
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| 19. |
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