In-vivo delivery of therapeutic proteins by genetically-modified cells: comparison of organoids and human serum albumin alginate-coated beads.
We have designed a self-assembling multimeric soluble CD4 molecule by inserting the C-terminal fragment of the alpha chain of human C4-binding protein (C4bp alpha) at the C-terminal end of human soluble CD4 genes. This CD4-C4bp alpha fusion protein (sMulti-CD4) and two other reference molecules, a fusion protein of human serum albumin (HSA) and the first two domains of CD4 (HSA-CD4) and monomeric soluble CD4 (sMono-CD4), were delivered in vivo by genetically modified 293 cells. These cells were implanted in mice as organoids and also encapsulated in HSA alginate-coated beads. sMulti-CD4 showed an apparent molecular weight of about 300-350 kDa, in accordance with a possible heptamer formula. sMulti-CD4 produced either in cell culture or in vivo in mice appeared to be a better invitro inhibitor of HIV infection than sMono-CD4. Plasma levels of sMulti-CD4, HSA-CD4, and sMono-CD4 reached approximately 2,300, 2,700, and 170 ng/mL, respectively, 13 weeks after in-vivo organoid implantation, which had formed tumours at that time. This suggests that the plasma half-life of sMulti-CD4 is much longer than that of sMono-CD4. The 293 xenogeneic cells encapsulated in HSA alginate-coated beads remained alive and kept secreting sMono-CD4 or HSA-CD4 continuously at significant levels for 18 weeks in nude mice, without tumour formation. When implanted in immunocompetent Balb/c mice, they were rejected two to three weeks after implantation. In contrast, encapsulated BL4 hybridoma cells remained alive and kept secreting BL4 anti-CD4 mAb for at least four weeks in Balb/c mice. These results suggest the clinical potential of the C4bp-multimerizing system, which could improve both the biological activity and the poor in-vivo pharmacokinetic performance of a monomeric functional protein like soluble CD4. These data also show that a systemic delivery of therapeutic proteins, including immunoglobulins, can be obtained by the in-vivo implantation of engineered allogeneic cells encapsulated in HSA alginate-coated beads.
Shinya
E
E
Laboratoire de biologie et thérapeutique des pathologies immunitaires, UPMC/CNRS ESA 7087, Hôpital de la Pitié-Salpêtrière, Paris, France.
Dervillez
X
X
Edwards-Lévy
F
F
Duret
V
V
Brisson
E
E
Ylisastigui
L
L
Lévy
M C
MC
Cohen
J H
JH
Klatzmann
D
D
D000464:Alginates; D000818:Animals; D001672:Biocompatible Materials; D015704:CD4 Antigens / Q000235:genetics*; D002214:Capsules; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D017690:Cell Transplantation; D003181:Complement C4 / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006801:Humans; D016167:Integrin alphaXbeta2; D007668:Kidney; D008297:Male; D051379:Mice; D008819:Mice, Nude; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012709:Serum Albumin / Q000235:genetics; D014162:Transfection; D014183:Transplantation, Heterologous
D000464:Alginates; D001672:Biocompatible Materials; D015704:CD4 Antigens; D002214:Capsules; D002352:Carrier Proteins; D003181:Complement C4; D006603:Hexuronic Acids; D016167:Integrin alphaXbeta2; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D020723:Glucuronic Acid