Half‐Life Extension of Therapeutic Proteins via Genetic Fusion to Recombinant PEG Mimetics

Binder, Uli; Skerra, Arne

doi:10.1002/9783527644827.ch4

Cited by 14 publications

(9 citation statements)

References 30 publications

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“…Second, synthetic polymers are typically not biodegradable, and the long-term effect of their in vivo accumulation is still not well understood; for example, PEG is known to accumulate in vacuoles in the kidney [7,11]. Third, although there have been significant advances in the synthesis of synthetic polymers with low polydispersity, [3] they are intrinsically polydisperse in their molecular weights (MW), which limits the ability to precisely tune their in vivo behavior [12][13][14]. Fourth, the poorly controlled stoichiometry and site specificity of most chemical conjugation approaches, as well as limited yield, also limit the utility of synthetic polymer conjugates for protein and peptide delivery [13,15].…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, recombinant peptide polymers, or polypeptides such as XTEN that do not have a discernible repeat unit, [16] can be attached to a peptide or protein drug with molecular precision, in terms of their sequence, stoichiometry, and chain length, as they are genetically encoded with the peptide or protein drug of interest [13,[17][18][19]. Their PK and tissue biodistribution are less variable due to their precise sequence and monodispersity, which gives this class of macromolecules a significant advantage over most synthetic alternatives that are polydisperse.…”

Section: Introductionmentioning

confidence: 99%

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Long circulating genetically encoded intrinsically disordered zwitterionic polypeptides for drug delivery

et al. 2019

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The clinical utility of many peptide and protein drugs is limited by their short in-vivo half-life. To address this limitation, we report a new class of polypeptide-based materials that have a long plasma circulation time. The design of these polypeptides is motivated by the hypothesis that incorporating a zwitterionic sequence, within an intrinsically disordered polypeptide motif, would impart "stealth" behavior to the polypeptide and increase its plasma residence time, a behavior akin to that of synthetic stealth polymers. We designed these zwitterionic polypeptides (ZIPPs) with a repetitive (VPX 1 X 2 G) n motif, where X 1 and X 2 are cationic and anionic amino acids, respectively, and n is the number of repeats. To test this hypothesis, we synthesized a set of ZIPPs with different pairs of cationic and anionic residues with varied chain length. We show that a combination of lysine and glutamic acid in the ZIPP confer superior pharmacokinetics, for both intravenous and subcutaneous administration, compared to uncharged control polypeptides.Finally, to demonstrate their clinical utility, we fused the best performing ZIPP sequence to glucagon-like peptide-1 (GLP1), a peptide drug used for treatment of type-2 diabetes and show Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supporting InformationSupporting Information is attached. The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Long circulating genetically encoded intrinsically disordered zwitterionic polypeptides for drug delivery

et al. 2019

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“…Another major advantage of PASylation is that the amino acid polymer can be genetically encoded and directly produced as a fusion protein, thus avoiding the laborious optimization of chemical coupling conditions. 37 Using premade gene cassettes, the precise tuning of the length of the PAS tag attached to a functional binding protein is easily achieved. Hence, the loss of binding activity, which often accompanies PEGylation of proteins, as well as the formation of a heterogeneous bioconjugate, is prevented.…”

Section: Introductionmentioning

confidence: 99%

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

Mendler

Friedrich

Laitinen

et al. 2015

mAbs

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Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

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“…Proteins with larger molecular size, in particular, PAS-Coversin with its diameter of 18.6 nm (vs Ø = 5.6 nm for plain Coversin), progressively resist glomerular filtration and, consequently, show longer plasma half-life . This is the first published application of PASylation technology to a member of the lipocalin protein family with therapeutic potential.…”

Section: Discussionmentioning

confidence: 90%

PASylated Coversin, a C5-Specific Complement Inhibitor with Extended Pharmacokinetics, Shows Enhanced Anti-Hemolytic Activity in Vitro

et al. 2016

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The Ornithodoros moubata Complement Inhibitor (OmCI) binds complement component 5 (C5) with high affinity and, thus, selectively prevents proteolytic activation of the terminal lytic complement pathway. A recombinant version of OmCI (also known as Coversin and rEV576) has proven efficacious in several animal models of complement-mediated diseases and successfully completed a phase Ia clinical trial. Coversin is a small 17 kDa lipocalin protein which has a very short plasma half-life if not bound to C5; therefore, the drug requires frequent dosing. We have improved the pharmacokinetics of Coversin by N-terminal translational conjugation with a 600 residue polypeptide composed of Pro, Ala, and Ser (PAS) residues. To this end, PAS-Coversin as well as the unmodified Coversin were functionally expressed in the cytoplasm of E. coli and purified to homogeneity. Both versions showed identical affinity to human C5, as determined by surface plasmon resonance measurements, and revealed similar complement inhibitory activity, as measured in ELISAs with human serum. In line with the PEG-like biophysical properties, PASylation dramatically prolonged the plasma half-life of uncomplexed Coversin by a factor ≥50 in mice. In a clinically relevant in vitro model of the complement-mediated disease paroxysmal nocturnal hemoglobinuria (PNH) both versions of Coversin effectively reduced erythrocyte lysis. Unexpectedly, while the IC values were comparable, PAS-Coversin reached a substantially lower plateau of residual lysis at saturating inhibitor concentrations. Taken together, our data demonstrate two clinically relevant improvements of PASylated Coversin: markedly increased plasma half-life and considerably reduced background hemolysis of erythrocytes with PNH-induced phenotype.

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Half‐Life Extension of Therapeutic Proteins via Genetic Fusion to Recombinant PEG Mimetics

Cited by 14 publications

References 30 publications

Long circulating genetically encoded intrinsically disordered zwitterionic polypeptides for drug delivery

Long circulating genetically encoded intrinsically disordered zwitterionic polypeptides for drug delivery

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

PASylated Coversin, a C5-Specific Complement Inhibitor with Extended Pharmacokinetics, Shows Enhanced Anti-Hemolytic Activity in Vitro

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