Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β.
OBJECTIVE
Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-β with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes.


METHODS
Mature cytokines cloned downstream of LAP and a MMP cleavage site were expressed in 293T cells and assessed for latency and biological activity by Western blotting and bioassay.


RESULTS
We demonstrate here that fusion proteins of TGF-β, erythropoietin, IL-1ra, IL-10, IL-4, BMP-7, IGF1 and IL-17 were rendered latent by fusion to LAP, requiring cleavage to become active in respective bioassays. As further proof of principle, we also show that delivery of engineered TGF-β can inhibit experimental autoimmune encephalomyelitis and that this approach can be used to efficiently deliver cytokines to the brain and spinal cord in mice with this disease.


CONCLUSIONS
The latent cytokine approach can be successfully applied to a range of molecules, including cytokines of different molecular structure and mass, growth factors and a cytokine antagonist.
William Harvey Research Institute Barts and The London Medical School, Bone and Joint Research Unit , Charterhouse Square, London WC1M 6BQ , UK +44 207 882 2352; +44 207 882 6121; y.chernajovsky@qmul.ac.uk.
Lisa
L
Mullen
Anne
A
Rigby
Michelle
M
Sclanders
Gill
G
Adams
Gayatri
G
Mittal
Julia
J
Colston
Rewas
R
Fatah
Cristina
C
Subang
Julie
J
Foster
Philippa
P
Francis-West
Mario
M
Köster
Hansjörg
H
Hauser
Lorna
L
Layward
Sandrine
S
Vessillier
Alex
A
Annenkov
Sarah
S
Al-Izki
Gareth
G
Pryce
Chris
C
Bolton
David
D
Baker
David J
DJ
Gould
Yuti
Y
Chernajovsky
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000188:drug therapy; D005347:Fibroblasts; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008811:Mice, Inbred DBA; D008907:Mink; D058990:Molecular Targeted Therapy; D010455:Peptides / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use
D016207:Cytokines; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; C472416:latency-associated propeptide, TGF-beta; D007334:Insulin-Like Growth Factor I; D020781:Matrix Metalloproteinase 1