DEVELOPMENT OF LATENT CYTOKINE FUSION PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 234

reported between the cells treated with intact LAP-MMP-
hsTNFR1 and MMP-treated LAP-MMP-hsTNFR1 are min-
imal. A much greater difference in biological activity
between the intact and cleaved LAP-fusion protein is
expected if the LAP-fusion protein is truly latent. An
example of the effect of MMP treatment on latent LAP-
MMP-IFN- b is shown in Figure 16.3c. Prior to MMP-1
treatment, the biological activity of the IFN- b in protecting
cells from viral infection is virtually absent. Treatment of
LAP-MMP-IFN- b with MMP-1 results in a dramatic
increase in biological activity (Figure 16.3c; also see [7]).
In addition, a monomeric soluble TNF-R1 has to be in great
excess to free TNF in order to properly antagonize. Hence,
we have designed the dTNFR that binds TNF in a stable one-
to-one molar interaction [27]. The molecular design
described by Xiong et al. [24] is not a viable TNF inhibitor.

followed by a MMP cleavage site, and then the therapeutic
cytokine of choice into a suitable expression vector.
One important point to note is that the LAP must be at the
N-terminus of the molecule, that is, N-terminal with regard to
the cytokine, as Adams et al. [7] have clearly shown that
cloning of the LAP at the C-terminus of the fusion protein fails
to confer complete latency. This is most likely due to the fact
that when dimerization of themolecules occur; the cytokine is
not enclosed within the LAP shell, and so retains the ability to
interact with its cellular receptors.
It is necessary to make two changes to the native LAP
sequence when cloning LAP for use in constructing latent
cytokines. The first is to remove the furin cleavage site at
position 278/279 of the LAP sequence, to prevent processing
of the latent cytokine by furin and consequent release of the
cytokine from the protective LAP shell. Hence, amino acids
Met 1 -Ser 273 of the LAP sequence are cloned. It is also
necessary to mutate the cysteine residue at position 33 in
the LAP sequence (Figure 16.4). This residue is involved in
binding of native LAP-TGF- b to a 125-210 kDa protein
known as latent TGF- b binding protein (LTBP), the function
of which is to ensure efficient secretion from the cell and to
target and tether latent TGF- b to the ECM, allowing subse-
quent activation of TGF- b [28,29,30]. Complexing of LAP

16.4 GENERATION OF LATENT CYTOKINES

16.4.1 Molecular Design

The cloning of recombinant latent cytokines is relatively
straightforward,

involving cloning the LAP of TGF- b ,

FIGURE 16.4 Structure of the latency-associated peptide (LAP) from TGF- b . (A) Schematic
representation of the native LAP structure. Printed from [30] with permission from the publisher.
Formation of disulfide bonds at Cys 223/225 results in dimerization of the LAP molecules to form a
shell surrounding the active cytokine. The native LAP/cytokine complex interacts with LTBP through
Cys 33 . (B) Amino acid sequence of human LAP indicating the location of the cysteine residues that
are important in binding of LAP to LTBP (Cys33) and in dimer formation (Cys 223 and Cys 225 ) in bold,
as well as the location of the RGD site. The locations of the heparin-binding sites are also shown, as
well as the glycine/serine linker and the MMP cleavage site. Source: (a) This research was originally
published in Reference [30].