FUSION PROTEIN LINKERS: EFFECTS ON PRODUCTION, BIOACTIVITY, AND PHARMACOKINETICS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 63

TABLE 4.3 Cleavable Linker Peptide Sequences Designed in the Fusion Proteins

Linker No. Sequence

Type 1 linkers 1 GGGGS- PLGLWA -GGGGS

2 GGGGS- GPLGLWA -GGGGS

3 GGGGS- GPLGLWAQ -GGGGS

Type 2 linkers 4 GGGGS- GVPDVG HF SLFP -GGGGS

5 GGGGS- GVPDVG EF SLFP -GGGGS

6 GGGGS- GVPDVG NF SLFP -GGGGS

7 GGGGS- GVPDVG RF SLFP -GGGGS

8 GGGGS- GVPDVG HY SLFP -GGGGS

9 GGGGS- GVPDVG EY SLFP -GGGGS

10 GGGGS- GVPDVG NY SLFP -GGGGS

11 GGGGS- GVPDVG RY SLFP -GGGGS

Type 3 linkers 12 GGGGS-NSG PLGLWA QSTSAGPTV-GGGGS

13 GGGGS-NS GVPDVGHFSLFP STSAGPTV-GGGGS

14 GGGGS-NS GVPDVGNYSLFP STSAGPTV-GGGGS

Different MMP sites are shown in bold. Underlined amino acids correspond to the positions X 1 and X 2 of the consensus
sequence Gly-Val-Pro-Asp-Val-Gly-X 1 X 2 -Ser-Leu-Phe-Pro based on the MMP prodomain region. Italics amino acids
correspond to the hydrophilic loop derived from the flanking sequences of the furin cleavage site from the anthrax
protective antigen.
Source: Reproduced from Reference [47] by permission of Oxford University Press.

acted as a latent cytokine. As a result, LAP-IFN- b fusion
protein had a long half-life of 55 h in vivo (37 times longer
than native IFN- b [53]), because the latent cytokine could
not interact with its cellular receptors until it was released
from the LAP. Conceivably, the side effect of IFN- b could
also be minimized because of the restricted exposure to the
released cytokine.
To obtain the optimal sensitivity of the linkers to the
MMPs, three different types of cleavage sites were intro-
duced into the LAP-IFN- b construct by Vessillier et al. [47]
(Table 4.3). The original MMP cleavage site (Pro-Leu-Gly-
Leu-Trp-Ala), derived from a fluorogenic substrate forMMPs
[54], was extended by addition of one or two amino acids (Gly
and Gln), to achieve a higher sensitivity to cleavage byMMPs
[55]. The second type of linker was based on cleavage sites
derived from the propeptide region of MMPs. A consensus
sequence Gly-Val-Pro-Asp-Val-Gly-X1X2-Ser-Leu-Phe-Pro
was identified based on the prodomain region of several
MMPs. Combinations of X1 (His, Glu, Asn, or Arg) and
X2 (Phe or Tyr) were used for the development of new MMP
cleavage sites. The last type of linkers added a surface-
exposed flexible loop, derived from the amino acids 162-
175 around the furin cleavage site of the anthrax toxin
protective antigen, to the MMPs cleavage site [56]. The
addition of this loop sequence was intended to increase the
accessibility of the MMP-sensitive sequence to the MMPs.
These cleavable linkers all exhibited various sensitivities
for the MMPs. Among them, the addition of the surface-
exposed flexible loop from the furin cleavage site of the
anthrax toxin protective antigen increased the sensitivity to
MMP-3 to up to 29-fold. The determination of cleavage
efficiency and specificity of the cleavage sites to the MMPs

could be useful for developing other biological agents that
could be activated at the disease sites overexpressing MMPs.
In addition to extracellular spaces, the activation site of
in vivo cleavable linkers in fusion proteins can also be
inside the cells. Another type of in vivo cleavable linker
takes advantage of furin, a cellular endoprotease that has
been implicated in the proteolytic activation of diverse
precursor proteins [57]. This endoprotease has the consensus
recognition sequence -Arg-X-Arg/Lys-Arg # -( # identifies the
cleavage site). Furin is mainly localized in trans-golgi net-
work (TGN) and also recycles between TGN, early endo-
somes, and the cell surface.
The furin-sensitive linkers have been applied in the
construction of recombinant fusion proteins such as immu-
notoxins or immunoproapoptotic proteins from which active
toxins or protein drugs can be released from scFv after
intracellular processing. Inclusion of furin-sensitive linkers
in immunotoxins containing ribotoxin, caspase-3, or gran-
zyme B has shown significant improvements in cytotoxicity
compared with constructs containing stable linkers [58,59].
Like the disulfide linker, the protease-sensitive linkers
can be applied to fusion proteins when the in vivo separation
of the domains is preferred. Additionally, using protease
sensitive linkers also offers a new approach to specifically
deliver prodrugs to target sites where the proteases that
convert the prodrugs are present.
In conclusion, various types of linkers with different
structures or functions have been reported for the construc-
tion of fusion proteins. As summarized in Table 4.4, each
type of linkers has its specific characteristics, advantages,
and disadvantages. The choice of linkers should be based on
the properties and applications of the desired fusion proteins.