FUSION PROTEIN LINKERS: EFFECTS ON PRODUCTION, BIOACTIVITY, AND PHARMACOKINETICS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 66

GH/
G-CSF

LE

Tf

Dipeptide (LE)

P R

S

F

F F

N

T

S
C

GH/
G-CSF

Cyclopeptide (cyclo)

Tf

S S

L E A G C G S L E

GH/
G-CSF

Helical peptide ((H4) 2 ) Tf

LEA(EAAAK) 4 ALEA(EAAAK) 4 ALE

FIGURE 4.3 Illustration of three linkers applied in GH-Tf and G-CSF-Tf fusion proteins for PK
study. The dipeptide linker LE is composed of two amino acids—Leu and Glu. The cyclopeptide
linker is derived from somatostatin, with a mutated thrombin-sensitive sequence “PRS.” Two
cysteine residues on the linkers naturally form a disulfide bond. The helical peptide linker adopts
an a -helical structure.

The helical linker may provide a relatively large space for
the domains to fold independently and properly. A correctly
folded fusion protein may have enhanced stability and
consequently, an increased expression level from the cells.

proteins, and also established a mechanistic model for PK
of fusion proteins.
To elucidate the role of linker insertion on PK of fusion
proteins, three linkers with different lengths and conforma-
tions (Figure 4.3) were inserted into bifunctional fusion
proteins composed of Tf and hGH or G-CSF. The first linker
is a short dipeptide, Leu-Glu (LE), resulting from the cloning
site XhoI between the two protein genes [13]. The second
linker is a thrombin-sensitive, disulfide cyclopeptide linker,
designated as cyclo. As described in Section 4.3.3, this
cyclopeptide linker with a medium length (20 amino acids)
and a rigid cyclic structure were originally designed to create
a disulfide linker that could be rapidly cleaved in vivo by
reduction [45]. The third linker is an a -helix forming linker,
designated as (H4) 2 (LEA(EAAAK) 4 ALEA(EAAA-
K) 4 ALE). This linker has the longest length among all the
three linkers (50 amino acids) and a rigid, extended structure
[14,26]. Previous reports suggest that this long helical linker
can spatially separate the functional domains and reduce the
steric hindrance between them [34].
After the insertion of three linkers, the receptor binding
affinities of hGH-Tf fusion proteins were first compared via
competitive receptor binding assays on receptor-bearing cell
lines. With the shortest dipeptide linker (LE), fusion protein
hGH-LE-Tf exhibited the lowest binding affinities for both
hGH receptor (hGHR) and Tf receptor (TfR) (Table 4.5),
suggesting that this short linker might generate strong steric
hindrance or cause interference between functional domains.
On the other hand, the cyclopeptide and helical peptide
linkers, which both have longer lengths (20 and 50 amino
acids, respectively) and more rigid structures, generated
higher receptor binding affinities (Table 4.5). Therefore, by
inserting different linkers between the functional domains, the
receptor binding affinities of bifunctional fusion proteins
could be greatly altered.

4.4.3 Linkers Can Affect the PK of Fusion Proteins

Besides their wide applications in biological and biomedical
research, recombinant bifunctional fusion proteins have
become an important class of therapeutics for the treatment
of human diseases. Several fusion proteins such as Enbrel 1
(TNF-R/Fc-IgG1), Ontak 1 (IL-2/diphtheria toxin), Orencia 1
(CTLA-4/Fc-IgG1), and Amevive 1 (LFA-3/Fc-IgG1) have
been approved by the FDA for clinical usage [65], and many
more are currently under development. Fusing two protein
domains together may provide many advantages over the
parent proteins. By fusing with HSA or the Fc portion of
IgG, many protein drugs such as insulin, IFN- a ,andTNFhave
exhibited improved PK and PD properties [62,66,67]. The
fusion proteins can also have bifunctional properties where
each domain imparts a bioactive property (e.g., human immu-
noglobulin Fc g Fc e bifunctional fusion protein) [68], or
where a drug targeting effect is combined with biological
activity (e.g., immunotoxins) [69].
In contrast to the rapid development of fusion proteins as
therapeutics, the PKs of bifunctional fusion proteins are still
largely unexplored due to their distinct bifunctional proper-
ties. Many bifunctional fusion proteins contain linkers
between functional domains to improve their bioactivity,
receptor binding affinity, as well as structural stability.
However, especially in the case of bifunctional fusion
proteins, linker insertion increases the complexity of the
PK studies and the impact of linkers on PK is a remaining
question. Recently, Chen et al. conducted a study demon-
strating that the linkers could affect the PK of fusion