DEVELOPMENT OF LATENT CYTOKINE FUSION PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 232

in activating the latent IFN- b , as opposed to sera from these
patients, which did not result in release of active IFN- b from
the latent complex. This indicates that the nature and
quantity of MMP in biological fluids in disease conditions
is sufficient to cleave the latent molecule and release the
active cytokine. Finally, comparison of the half-lives of the
latent IFN- b and free IFN- b showed a highly significant
(37-fold) increase in the half-life of the latent cytokine
compared with the free cytokine [7]. The half-life of the
latent IFN- b is also greater than that of pegylated IFN- b
[22], which is a commonly used method of extending the
half-life of therapeutic proteins.
One of the main strengths of this technology is that
targeting of the therapeutic cytokine to the sites of disease
is achieved without having to identify specific disease anti-
gens, as is the case when using antibody-cytokine fusions.
Another important aspect of the latent approach is that
because the LAP is a native protein, the immunogenicity
of the latent complex should be virtually nonexistent, and
there are fewer problems with interactions with immune cells
that are characteristic of antibody Fc fusions. Neither of these
types of fusion proteins with immunoglobulin fragments or
pegylation confers latency. Hence, the latent cytokine tech-
nology is applicable to a much wider range of pathological
conditions and causes fewer side effects.

TABLE 16.1 Human Matrix Metalloproteinases (MMPs)

Name MMP Location

Collagenases
Collagenase 1

MMP-1 Secreted

Collagenase 2 MMP-8 Secreted

Collagenase 3 MMP-13 Secreted

Gelatinases
Gelatinase A

MMP-2 Secreted

Gelatinase B MMP-9 Secreted

Stromelysins
Stromelysin 1 MMP-3 Secreted

Stromelysin 2 MMP-10 Secreted

Matrilysins
Matrilysin 1 MMP-7 Secreted

Matrilysin 2 MMP-26 Secreted

Stromelysin 3 MMP-11 Secreted

Membrane-type MMPs
MT1-MMP MMP-14 Membrane associated

MT2-MMP MMP-15 Membrane associated

MT3-MMP MMP-16 Membrane associated

MT4-MMP MMP-17 Membrane associated

MT5-MMP MMP-24 Membrane associated

MT6-MMP MMP-25 Membrane associated

Others
Macrophage elastase

MMP-12 Secreted

MMP-19 Secreted

Enamelysin MMP-20 Secreted

16.3 LIMITATIONS OF THE LATENT CYTOKINE
TECHNOLOGY

MMP-21 Secreted

CA-MMP MMP-23 Secreted

MMP-27 Secreted

Epilysin MMP-28 Secreted

The latent cytokine technology was initially designed to
deliver therapeutic cytokines to the sites of disease. How-
ever, we have also used this technology to produce cytokine
antagonists using receptors for pro-inflammatory cytokines
inside the LAP shell as anti-inflammatory therapeutics.
These efforts have focused in particular on the engineering
of a dimeric TNF receptor 2 (p75) (dTNFR) [23] inside the
LAP shell, as the central role of TNF- a in initiating and
maintaining inflammation is well established. It was hypoth-
esized that this LAP-MMP-dTNFR protein could be used to
block TNF- a activity in a manner similar to that achieved by
the anti-TNF- a antibodies used in the treatment of RA
patients [6], with the possible added advantage of activity
only at sites of disease. To test this hypothesis, LAP-MMP-
dTNFR was expressed in 293 T cells. Cell supernatants
containing the recombinant protein were treated with MMP-
1, followed by SDS-PAGE and Western blotting using both
anti-LAP and anti-TNFR antibodies. These experiments
showed the presence of recombinant protein of the expected
size, and after treatment with MMP-1, the release of indi-
vidual components of TNFR and LAP (Figure 16.3a).
Subsequently, experiments to examine the biological activ-
ity of LAP-MMP-dTNFR were performed, using HeLa cells
that respond to hTNF stimulation with luciferase expression

providing a means by which a more severe disease pheno-
type will result in the release of more of the therapeutic
cytokine from its latent form.
The effectiveness of latent cytokines in treating disease has
been demonstrated by Adams et al. [7] using latent IFN- b to
treat collagen-induced arthritis (CIA) in mice, a well-known
mousemodel of RA. It had previously been shown that IFN- b ,
delivered as gene therapy had therapeutic effects in CIA [21]
so a plasmid encoding the latent IFN- b (including the MMP
cleavage site) was delivered via a single intramuscular injec-
tion into mice with CIA. The latent construct resulted in
inhibition of paw swelling and reduced clinical score [7].
This study also demonstrated three very important
aspects of the latent cytokine technology. First, the latent
IFN- b was biologically inactive until such time as the
molecule was treated with MMP to remove the LAP part
of the molecule and release the IFN- b . This result shows that
true latency can be achieved, even when the cytokine inside
the LAP shell is bigger in size than the native TGF- b .
Second, synovial fluid from arthritic joints, or cerebral
spinal fluid from meningitis or MS patients, was effective