Id |
Subject |
Object |
Predicate |
Lexical cue |
T87 |
0-2 |
Sentence |
denotes |
3. |
T88 |
3-13 |
Sentence |
denotes |
Discussion |
T89 |
14-214 |
Sentence |
denotes |
The present data demonstrate that the PASylation technology can solve two major problems of a peptide drug: (i) instability in the expression host and (ii) low bioactivity due to fast renal clearance. |
T90 |
215-456 |
Sentence |
denotes |
Typically, small peptides, if expressed in a soluble state in E. coli, are quickly degraded by host cell proteases [42] and, thus, require production as larger fusion proteins as well as subsequent release by site-specific cleavage in vitro. |
T91 |
457-699 |
Sentence |
denotes |
Here, fusion with a random-coil forming PAS sequence of ~600 amino acids prevented proteolytic degradation and allowed cost-efficient one-step production of the intact fusion protein in E. coli without the need of additional processing steps. |
T92 |
700-1126 |
Sentence |
denotes |
Of note, in contrast to classical approaches, which involve the use of an insoluble fusion partner, for example, an α-galactosidase fragment in the case of insulin [43], to provoke formation of inclusion bodies and protect the gene product from intracellular proteolysis, the PASylated Tα1 peptide was recovered as a soluble fusion protein, thus allowing direct purification from the cell extract without solubilization steps. |
T93 |
1127-1251 |
Sentence |
denotes |
Furthermore, the C-terminally attached PAS moiety was compatible with N-terminal acetylation by RimJ as confirmed by ESI-MS. |
T94 |
1252-1580 |
Sentence |
denotes |
Yields of our expression study at the research scale reached 15 mg purified acetylated Tα1-PAS and even 50 mg PAS-Tα1 per 1 L bacterial culture (at an optical density (OD) of 3), which surpasses the reported yield of a bacterially produced (yet probably non-acetylated and non-glycosylated) Tα1-Fc fusion protein (16 mg/L) [44]. |
T95 |
1581-1822 |
Sentence |
denotes |
Still, there is considerable opportunity of improvement for both versions of the PASylated peptide by optimizing the expression plasmid and the production process, including high cell density fermentation under controlled feeding conditions. |
T96 |
1823-2014 |
Sentence |
denotes |
Apart from that, the absence of a prominent band for the co-expressed enzyme RimJ in SDS-PAGE despite approximately 50% N-terminal acetylation of Tα1-PAS also indicates room for amelioration. |
T97 |
2015-2245 |
Sentence |
denotes |
Optimization of the ribosome-binding site preceding the RimJ cistron or changing the order within the bicistronic operon should boost biosynthesis of the enzyme and result in increased yield of the N-terminally acetylated Tα1-PAS. |
T98 |
2246-2511 |
Sentence |
denotes |
Finally, the use of a high-efficiency secretory bacterial expression system such as ESETEC [45] or CORYNEX [46,47] should lead to higher product titers as previously demonstrated for other PASylated fusion proteins such as PASylated human growth hormone (hGH) [48]. |
T99 |
2512-2691 |
Sentence |
denotes |
The yield of functional PAS-hGH was more than 100-fold higher with the ESETEC system than in a conventional laboratory strain of E. coli, reaching several grams per liter culture. |
T100 |
2692-2841 |
Sentence |
denotes |
Attachment of the PAS sequence at either end of the Tα1 peptide increased the hydrodynamic volume by more than an order of magnitude as shown by SEC. |
T101 |
2842-3068 |
Sentence |
denotes |
This strongly expanded molecular size resulted in a plasma half-life in rats of around 16 h after a subcutaneous injection of Tα1-PAS, which is more than an 8-fold increase compared to the unmodified peptide drug (1.9 h) [41]. |
T102 |
3069-3266 |
Sentence |
denotes |
Based on in vitro cell culture assays with human serum albumin fusion proteins [49], both the C-terminus and the N-terminus of Tα1 should be permissible to modification while retaining bioactivity. |
T103 |
3267-3457 |
Sentence |
denotes |
This was also shown in animal tumor models for C-terminal fusion with an immunoglobulin Fc fragment [41], an internalizing arginylglycylaspartic acid peptide iRGD [50], and thymopentin [51]. |
T104 |
3458-3705 |
Sentence |
denotes |
However, in the case of Tα1, some caution is appropriate regarding the significance of in vitro cell culture assays as its mode of action is complex and involves different receptors provoking multiple biological effects on various cell types [52]. |
T105 |
3706-3898 |
Sentence |
denotes |
Second, a prolonged circulation in the body as demonstrated here via application of the PASylation technology influences both binding kinetics and bioactivity, which is not reflected in vitro. |
T106 |
3899-4273 |
Sentence |
denotes |
While attachment of large macromolecules, such as PAS polypeptides or albumin, but also PEG [53], can lead to lower receptor association rates for bioactive peptides or proteins, this is usually overcompensated by the drastically prolonged in vivo half-life, which results in a strongly enhanced bioactivity as demonstrated for multiple PASylated biopharmaceuticals [37,54]. |
T107 |
4274-4432 |
Sentence |
denotes |
In the case of Tα1, superior effects in preclinical cancer models due to prolonged circulation were recently demonstrated for a Tα1-Fc fusion protein [41,44]. |
T108 |
4433-4648 |
Sentence |
denotes |
Such studies would be the obvious next step to investigate enhanced in vivo bioactivity of PASylated Tα1, and it will be interesting to see whether its N-terminally or C-terminally PASylated version performs better. |
T109 |
4649-4944 |
Sentence |
denotes |
In comparison with other published approaches to prolong the circulation of Tα1, the measured terminal half-life of Tα1-PAS is even longer than the value of around 8.2 h reported for a corresponding PEGylated peptide after an intravenous injection into the tail vein of Sprague–Dawley rats [55]. |
T110 |
4945-5085 |
Sentence |
denotes |
This modified Tα1 was prepared by chemically coupling a 5 kDa methoxypolyethylene glycol maleimide via an engineered N-terminal Cys residue. |
T111 |
5086-5322 |
Sentence |
denotes |
Of note, according to the rules of allometric scaling, a much longer half-life in the range of several days can be expected for Tα1-PAS in humans [56,57], which would allow weekly dosing while achieving a lasting pharmacological effect. |
T112 |
5323-5567 |
Sentence |
denotes |
According to the prescription information for Zadaxin™ to treat chronic hepatitis B, the recommended dose for the monotherapy, or the combination therapy with interferon, is a 1.6 mg subcutaneous injection twice weekly over 6 months (52 doses). |
T113 |
5568-5678 |
Sentence |
denotes |
Consequently, weekly or biweekly injections would considerably decrease patient burden and improve compliance. |
T114 |
5679-5893 |
Sentence |
denotes |
Moreover, a more continuous plasma level above the minimum effective dose due to slower clearance, as illustrated by the drastically increased AUC, should boost in vivo efficacy and open new treatment perspectives. |
T115 |
5894-6282 |
Sentence |
denotes |
Especially in preclinical animal models, which typically suffer from a much quicker drug clearance compared to humans [37] owing to their smaller body size, a long-acting Tα1 should lead to more convincing pharmacodynamic (PD) effects and pave the way for biopharmaceutical development for novel indications such as cystic fibrosis [18], HIV-1 infection [58], sepsis [20], or cancer [59]. |
T116 |
6283-6421 |
Sentence |
denotes |
For example, cancer studies have demonstrated that high doses of the conventional peptide are required to achieve antitumor activity [60]. |
T117 |
6422-6541 |
Sentence |
denotes |
The beneficial application of the PASylation technology demonstrated in this work can be transferred to other peptides. |
T118 |
6542-6757 |
Sentence |
denotes |
There are more than 7000 naturally occurring peptides covering a wide range of physiological functions [61], including many peptides with proven therapeutic potential, which could profit from the presented approach. |
T119 |
6758-6987 |
Sentence |
denotes |
Examples are therapeutically active peptides such as thymosin beta 4 [62,63], the C-type natriuretic peptide (CNP) [64], human parathyroid hormone (PTH) [65], relaxin [66], or glucagon-like peptide-1 (GLP-1) and its analogs [67]. |
T120 |
6988-7215 |
Sentence |
denotes |
Today, it is generally recognized that intrinsic weaknesses of this drug class, such as poor stability and short circulating plasma half-life, need to be addressed in order to transform peptides into efficacious medicines [61]. |
T121 |
7216-7324 |
Sentence |
denotes |
The approach described here, N- or C-terminal PASylation, optionally combined with acetylation, solves both. |
T122 |
7325-7523 |
Sentence |
denotes |
On the one hand, N-terminal acetylation protects peptides from proteolytic degradation by exoproteases, for example, dipeptidyl peptidase-4 (DPP-IV), as shown for N-terminally acetylated GLP-1 [68]. |
T123 |
7524-7763 |
Sentence |
denotes |
On the other hand, PASylation increases the hydrodynamic volume of the peptide above the pore size of the glomerular basement membrane, hence retarding kidney filtration and prolonging the pharmacodynamic effect of its fusion partner [31]. |
T124 |
7764-7836 |
Sentence |
denotes |
Furthermore, PASylation can serve as a linker to join two peptides [69]. |
T125 |
7837-7961 |
Sentence |
denotes |
This is of particular interest if both entities act synergistically, for example, GLP-1 and GIP [70] or Tα1 and GM-CSF [71]. |
T126 |
7962-8227 |
Sentence |
denotes |
Alternatively, the biologically active protein/peptide can be linked via the PAS sequence to a targeting domain such as the arginylglycylaspartic acid peptide RGD peptide which binds to integrins αVβ3 and αVβ5 in order to enhance tumor penetration and accumulation. |
T127 |
8228-8385 |
Sentence |
denotes |
In fact, fusion of the short-acting Tα1 with the tumor-targeting iRGD peptide using a Gly4 linker has recently demonstrated enhanced antitumor activity [50]. |
T128 |
8386-8558 |
Sentence |
denotes |
In contrast to synthetic PEG linkers, which are commonly used for bioconjugations, the recombinant PAS sequence exhibits a precisely defined size and is biodegradable [72]. |
T129 |
8559-8738 |
Sentence |
denotes |
The PAS polypeptide itself is stable in blood plasma but quickly degraded by intracellular enzymes, thus avoiding organ accumulation [31], a well-known effect for PEGylation [73]. |
T130 |
8739-9056 |
Sentence |
denotes |
Moreover, PAS sequences are non-immunogenic in animals [31,74] and offer a one-step production of PASylated peptides in various commercially scalable expression systems including bacteria, yeasts, or mammalian cells [35,48], which would even allow the preparation of peptides carrying posttranslational modifications. |