FUSION PROTEINS FOR HALF-LIFE EXTENSION - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 97

Th e success of natural peptide repe ats to extend prot ein
half-lives in plas ma has stim ulated research to engi neer
artificial sequen ces that could even be super ior. Initially,
single amino aci d repe ats utilizi ng polyglyc ine have been
tried becau se an unstruct ured confo rmation could be
expecte d from in silico simulations . However, long glyc ine
polymers suffer from a low solubil ity in water, theref ore the
hydrophilic amino acid serine was introdu ced. The typical
(glycine 4 serine) n repe at has been used in many linker
sequences betwee n two domains before. Th is pent amer
was multipl ied either 20- or 40 -fold to gener ate a 100 or
200 residue polymer that was fused to the C-te rminus of a
light chain in a Fab fragme nt. The Fab maintai ned it s affinity
to its target and showed a threefold incr eased half-life for the
200 residue s polymer. Wit h size exclusion chromatogr aphy
the expansion of its appar ent siz e by 120% could be dem-
onstrated, although the real mass increase in comparison to
the origina l Fab was only 29%. Larger polymers are difficul t
to gener ate, first due to some limitations of the genet ic
construct but mainly becau se of the tendency to aggregate
at higher copy numb ers. Nevertheles s the threefold half-life
increase could be suffic ient for in vivo imaging [62] .
In an attempt to overco me the obst acles of this polymer, it
was tested if the addition of proline to the hydrop hilic amino
acid serine and a sma ll aliphati c one like ala nine instea d of
glycine could suppress the tend ency to aggregate. The
constant variat ion of the residue s witho ut long stretches
of iden tical amino acids should also reduc e the potential to
form preferred helica l or sheet-li ke conform ations. Besides
the highe r degree of variabilit y, through a third amino acid,
proline als o adds the abili ty for cis/tran s isomer ization that
gives further options for conform ational flexibilit y. Large
so-called PAS- sequences result in a dras tically incr eased
hydrodynam ic radius becau se o f the lack of stru ctural
conformat ion, and should also incr ease in vivo half-life of
proteins fused to this PAS-poly mer. Intere stingly, the PAS
polymer does neither contain mot ifs allowing the cleavage

by seru m prot eases, nor T-cell epitopes. PASylat ion 1 ,as
this technol ogy is calle d, is current ly tested with a
number of therape utic protei ns (ht tp://www.xl- protein.com /
technol ogy.html).
A simi lar appro ach to artificiall y engineer a pept ide
polymer with random confo rmation start ed with a bigge r
set of suitabl e amino acids (alanine, glyc ine, glut amate,
proline, serine, and threoni ne), excluding larger, hydrop ho-
bic, positively charged, or sulfur-containing amino acids.
These residue s wer e system atically screened to find combi-
nations that ful filled the criteri a of being unst ructured and
nonrepet itive. The selected candi date seque nce cal led
XTEN 1 contained 864 amino aci ds and extended the
half-life of Exenatide in mice by a factor of 71. Dur ing
size exclusion chromat ography (SEC) protei ns with the
attached XTEN seque nce eluted muc h earlier from the
column than wha t coul d be expected by their no minal
molecu lar mass, thus indicating a significan tly increased
hydrod ynamic radius [63]. The in vivo half-lif e extension is
probably als o influe nced by the high negative net-cha rge
contributed by a high glut amate cont ent that can cause
charge repulsi on at the glomer uli. This high negative
charge lowers the isoe lectric al point, pI, which coul d be
benefic ial for chromat ographic purifi cations based on ion
exchange, but could cause problems when addre ssing mol-
ecules on negatively charged cell surf aces. When tes ting a
glucagon-XTEN fusion protein, it was found that the potency
was only 15% of glucagon without XTEN. The reduction of
potency was inde pendent of t he length of the X TEN poly-
mer. But the polymer increased solubilit y 60-fold [64].
Ta bl e 6 .4 summari zes a ll peptid e-based half- life extension
st ra teg ies .

6.3.2 Glycos ylated Pe ptides

The hydrody namic radius can als o be increas ed by attac hing
carbohydr ates. An early example is a hyperglycosylated

TABLE 6.4 Peptide Fusion Based Half-Life Extension Strategies

Name Amino Acid Composition References/Websites

Large molecule binding Albumin-binding peptide RLIEDICLPRWGCLWEDD [48]

Soluble peptide polymer Antigen 13 repeats [EPSKA] n [60]

Deimmunized antigen 13 [PSTAD] n [61]

Homo-amino-acid polymer [G 4 S] n [62]

PASylation P, A, S http://www.xl-protein.com

XTENylation P, A, S, E, T, G [63,64]

Glycosylation C-terminal peptide [SSSKAPPPSLPSPSRLPGPSDTPILPQ] n [66]

Hydroxy proline glycosylation [S/AHypS/A/Hyp] n or [SHyp4] n [69,70]

Genetic polymers G, N, Q and A, S, T, D, E http://www.aequusbiopharma.com

[VPGX a G] n

Aggregate forming Elastin-like peptide http://www.phasebio.com

[GX b Z] n Gelatin-like peptide [74]

Hyp, hydroxyproline; X a , any amino acid except P; X b ,E,K,N,P,Q,orS;Z,X b except S.