Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life

Schlapschy, Martin; Theobald, Ina; Mack, Hildegard I. D.; Schottelius, Margret; Wester, Hans‐Jürgen; Skerra, Arne

doi:10.1093/protein/gzm020

Cited by 94 publications

(69 citation statements)

References 58 publications

(82 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Furthermore, binding of scDb and scDb-ABD to these cell lines was not affected in the presence of serum albumin. This finding is in accordance with results obtained for a half-life-extended anti-HER2 Fab 4D5, which was either fused to the same ABD or an albumin-binding peptide (AB.Fab4D5) (12,13). In contrast, different results were described for PEGylated antibody fragments.…”

Section: Discussionsupporting

confidence: 90%

Biodistribution of a Bispecific Single-chain Diabody and Its Half-life Extended Derivatives

Stork

Campigna

Robert

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Small recombinant antibody molecules such as bispecific single-chain diabodies (scDb) possessing a molecular mass of ϳ55 kDa are rapidly cleared from circulation. We have recently extended the plasma half-life of scDb applying various strategies including PEGylation, N-glycosylation and fusion to an albumin-binding domain (ABD) from streptococcal protein G. Here, we further analyzed the influence of these modifications on the biodistribution of a scDb directed against carcinoembryonic antigen (CEA) and CD3 capable of retargeting T cells to CEAexpressing tumor cells. We show that a prolonged circulation time results in an increased accumulation in CEA ؉ tumors, which was most pronounced for scDb-ABD and PEGylated scDb. Interestingly, tumor accumulation of the scDb-ABD fusion protein was ϳ2-fold higher compared with PEGylated scDb, although both molecules exhibit similar plasma half-lives and similar affinities for CEA. Comparing half-lives in neonatal Fc receptor (FcRn) wild-type and FcRn heavy chain knock-out mice the contribution of the FcRn to the long plasma half-life of scDb-ABD was confirmed. The half-life of scDb-ABD was ϳ2-fold lower in the knock-out mice, while no differences were observed for PEGylated scDb. Binding of the scDb derivatives to target and effector cells was not or only marginally affected by the modifications, although, compared with scDb, a reduced cytotoxic activity was observed for scDb-ABD, which was further reduced in the presence of albumin. In summary, these findings demonstrate that the extended half-life of a bispecific scDb translates into improved accumulation in antigen-positive tumors but that modifications might also affect scDb-mediated cytotoxicity.Bispecific single-chain diabodies (scDb) 2 are recombinant molecules composed of the variable heavy and light chain domains of two antibodies connected by three linkers in the order V H A-V L B-V H B-V L A (1). These domains assemble into molecules with a compact structure and molecular masses of ϳ55 kDa. Bispecific single-chain diabodies have been developed for various applications including the retargeting of cytotoxic T lymphocytes to tumor cells for cellular cancer therapy (2).Although scDb are capable of efficiently retargeting effector cells to tumor cells the small size leads to their rapid elimination after i.v. injection. The terminal half-life of these molecules in mice is only in the range of 5-6 h, compared with several days for whole IgG molecules (3, 4). The fast clearance of such small molecules from circulation hampers therapeutic applications, e.g. requiring infusions or repeated injections to maintain a therapeutically effective dose over a prolonged period of time (5). For example, a bispecific tandem scFv directed against CD19 and CD3 (blinatumomab) having a similar size as an scDb molecule had to be given as an 8-week infusion (maximum dose 60 g/m 2 per day) in a clinical phase I trial for the treatment of B cell lymphoma patients (6).To extend plasma half-lives of therapeutic proteins and thus to improve ph...

show abstract

Section: Discussionsupporting

confidence: 90%

Biodistribution of a Bispecific Single-chain Diabody and Its Half-life Extended Derivatives

Stork

Campigna

Robert

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The synthetic genes for most PAS sequences used in this study were either obtained by ligation of short hybridized oligodeoxynucleotide pairs encoding repetitive amino acid sequence units according to a previously described strategy18 or as assembled longer synthetic DNA fragments with minimized repeat on the nucleotide sequence level 8. The PAS polypeptides were produced in the cytoplasm of E. coli KS272 as C‐terminal fusion proteins with E. coli thioredoxin (TrxA; UniProt ID: P0AA25).…”

Section: Methodsmentioning

confidence: 99%

The polypeptide biophysics of proline/alanine‐rich sequences (PAS): Recombinant biopolymers with PEG‐like properties

Breibeck

Skerra

2017

Biopolymers

View full text Add to dashboard Cite

PAS polypeptides comprise long repetitive sequences of the small L‐amino acids proline, alanine and/or serine that were developed to expand the hydrodynamic volume of conjugated pharmaceuticals and prolong their plasma half‐life by retarding kidney filtration. Here, we have characterized the polymer properties both of the free polypeptides and in fusion with the biopharmaceutical IL‐1Ra. Data from size exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy and quantification of hydrodynamic and polar properties demonstrate that the biosynthetic PAS polypeptides exhibit random coil behavior in aqueous solution astonishingly similar to the chemical polymer poly‐ethylene glycol (PEG). The solvent‐exposed PAS peptide groups, in the absence of secondary structure, account for strong hydrophilicity, with negligible contribution by the Ser side chains. Notably, PAS polypeptides exceed PEG of comparable molecular mass in hydrophilicity and hydrodynamic volume while exhibiting lower viscosity. Their uniform monodisperse composition as genetically encoded polymers and their biological nature, offering biodegradability, render PAS polypeptides a promising PEG mimetic for biopharmaceutical applications.

show abstract

“…They also showed that the protein tags with the most disorder can be used not only for the increase in the solubility of target proteins as the aforementioned entropic bristles do, but also can serve as important tools for extending half-life of proteins and for characterizing their biological properties, e.g., binding. 129 An incomplete list of the disordered serine-containing tags shown to increase the half-life of a protein of interest inside the cell includes an unstructured recombinant polypeptide of 864 amino acids with the PESTAG composition, called XTEN, 130,131 serine-glycine-containing HAP 132 and proline-alanine-serine-containing PAS tags, 133 as well as tags containing PESAK, PASTDH, and PASTD repeats. 134,135 Some functions of serine-rich proteins To get a clue on what proteins with high serine content can be used for biologically, functions of several proteins with polyserine regions are outlined below.…”

Section: Serine and Functions Of Idps/idprsmentioning

confidence: 99%

The intrinsic disorder alphabet. III. Dual personality of serine

Uversky

2015

Intrinsically Disordered Proteins

View full text Add to dashboard Cite

Proteins are natural polypeptides consisting of 20 major amino acid residues, content and order of which in a given amino acid sequence defines the ability of a related protein to fold into unique functional state or to stay intrinsically disordered. Amino acid sequences code for both foldable (ordered) proteins/domains and for intrinsically disordered proteins (IDPs) and IDP regions (IDPRs), but these sequence codes are dramatically different. This difference starts with a very general property of the corresponding amino acid sequences, namely, their compositions. IDPs/IDPRs are enriched in specific disorder-promoting residues, whereas amino acid sequences of ordered proteins/domains typically contain more order-promoting residues. Therefore, the relative abundances of various amino acids in ordered and disordered proteins can be used to scale amino acids according to their disorder promoting potentials. This review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and represents serine, which is the third most disorder-promoting residue. Similar to previous publications, this review represents some physico-chemical properties of serine and the roles of this residue in structures and functions of ordered proteins, describes major posttranslational modifications tailored to serine, and finally gives an overview of roles of serine in structure and functions of intrinsically disordered proteins.

show abstract

Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life

Cited by 94 publications

References 58 publications

Biodistribution of a Bispecific Single-chain Diabody and Its Half-life Extended Derivatives

Biodistribution of a Bispecific Single-chain Diabody and Its Half-life Extended Derivatives

The polypeptide biophysics of proline/alanine‐rich sequences (PAS): Recombinant biopolymers with PEG‐like properties

The intrinsic disorder alphabet. III. Dual personality of serine

Contact Info

Product

Resources

About