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 (C4bpα) at the C-terminal end of human soluble CD4 genes. This CD4- C4bpα 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 BLA 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.
- Gene therapy