FUSION PROTEIN LINKERS: EFFECTS ON PRODUCTION, BIOACTIVITY, AND PHARMACOKINETICS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 64

TABLE 4.4 Summary of Empirical Linkers

Flexible Linker Rigid Linker Cleavable Linker

Characteristics Rich in small or hydrophilic aa Rigid structure or rich in Pro Cleavable by reduction or proteases

Examples (GGGGS) n ,G 8 (EAAAK) n , (XP) n Disulfide, protease-sensitive sequence

Advantages Flexibility and mobility Effective separation Release of free domain at specific site

Disadvantages Insufficient separation Low flexibility Possible low sensitivity to protease

4.4 FUNCTIONALITY OF LINKERS
IN FUSION PROTEINS

inserting a flexible (GGGGS) 3 linker between the two
functional domains [14]. All of the fusion proteins with
either helical or flexible linkers exhibited improved in vitro
G-CSF biological activities compared to the original fusion
protein with the dipeptide linker. Particularly, the fusion
protein with the (H4) 2 linker, (A(EAAAK) 4 ALEA-
(EAAAK) 4 A) exhibited the highest in vitro biological activ-
ity among all of the linkers tested, with approximately a 10-
fold increase in activity compared to the fusion protein with
the dipeptide linker. With the (H4) 2 linker improved in vivo
biological activities of G-CSF-Tf were also observed in
animal experiments viaboth subcutaneous andoral adminis-
tration. The subcutaneously administered G-CSF-(H4) 2 -Tf
proteinexhibitedmuchhigher efficacy thanG-CSF-LE-Tf at
three different doses (0.1, 0.5, and 1mg/kg). At a lowdose of
20mg/kg for oral administration, the G-CSF-(H4) 2 -Tf
fusion proteins elicited a significant pharmacological effect,
whereas the original fusion protein with the dipeptide linker
only exhibited minimal effect.
The insertion of the helical linker (H4) 2 had a similar
enhancement effect on the bioactivity of another Tf-based
fusion protein—human growth hormone (hGH)-Tf fusion
protein [26]. The in vitro hGH biological activity in hGH-
(H4) 2 -Tf was improved compared to hGH-Tf which contains
a short dipeptide linker LE. The in vivo bioactivity was then
tested in hypophysectomized rats, which gain body weight in
response to hGH treatment. The subcutaneous administration
of hGH-(H4) 2 -Tf promoted higher bodyweight gain than that
of hGH-Tf in a 7-day daily treatment. More remarkably, with
the oral administration, only hGH-(H4) 2 -Tf, but not hGH-Tf,
elicited a significant body weight gain [26].
These studies suggest that linker insertion may be a
useful method to improve suboptimal intrinsic activity of
fusion proteins that are caused by insufficient separation of
functional domains. The unwanted interaction and steric
hindrance between the protein domains could be greatly
decreased, and as a result, the bioactivity may be greatly
enhanced. In the study of G-CSF-Tf fusion proteins, the
helical peptide linkers appear to be superior to the flexible
linker (GGGGS) 3 , probably because the helical linkers
adopt more rigid structures and can separate the protein
domains more effectively.
Linker engineering was also applied to the construction
of bioactive and stable fusion proteins consisting of human

As previously discussed, various types of linkers are avail-
able for the purpose of protein fusion. They can have
different lengths, exhibit various conformations, and
even be designed for cleavage at specific sites in vivo.
These diverse characteristics of linkers provide them a
wide spectrum of functions. Besides the properties such
as covalently joining domains (e.g., V H and V L of anti-
body), spatially separating protein moieties, linkers can
also offer many advantages in improving the biological
activities, increasing production, as well as achieving desir-
able PD and PK properties of the fusion proteins.

4.4.1 Linkers Can Improve Biological Activity
of Fusion Proteins

A common issue encountered during the development of
fusion protein is the impaired biological activity of the
functional domains after protein fusion. One possible reason
for this issue is that the two functional domains are brought too
close, so that they can interfere with each other and block the
accessibility of the protein domains to the respective recep-
tors. To construct bioactive fusion proteins, linkers have been
very effective tools to provide enough distance between
domains and reduce the steric hindrance between them.
A good example of applying linkers for bioactivity
improvement is the development of Tf fusion proteins for
oral delivery of protein and peptide drugs [13,14,26]. The
Tf-fusion protein can be orally absorbed across intestinal
epithelium cells via transcytosis after binding to Tf receptors
on the intestinal cell surface [60,61]. AG-CSF and Tf fusion
protein with a short dipeptide linker (Leu-Glu, LE) was
constructed for oral delivery of G-CSF [13]. With the
dipeptide linker, the G-CSF-LE-Tf fusion protein elicited
significant pharmacological affect via oral administration at
a high dose of 50 mg/kg. However, the intrinsic G-CSF
bioactivity, as determined by in vitro cell assay, was sub-
optimal. The fusion protein retained less than 10% of the
bioactivity of parent G-CSF. To achieve greater oral efficacy,
Bai and Shen reported the construction of G-CSF-Tf fusion
proteins by inserting different copies of helical peptide
linker

[A(EAAAK) n A] m (n ¼ 2-4, m ¼ 1 or 2), or by