Synthetic HLA-G proteins for therapeutic use in transplantation

Abstract

The human leukocyte antigen (HLA)-G is a tolerogenic molecule, whose expression by allografts is associated with better acceptance. An increasing interest in producing HLA-G as a clinical-grade molecule for therapy use is impaired by its complexity and limited stability. Our purpose was to engineer simpler and more stable HLA-G-derived molecules than the full-length HLA-G trimolecular complex that are also tolerogenic, functional as soluble molecules, and compatible with good manufacturing practice (GMP) production conditions. We present two synthetic molecules: (α3-L)x2 and (α1-α3)x2 polypeptides. We show their capability to bind the HLA-G receptor LILRB2 and their functions in vitro and in vivo. The (α1-α3)x2 polypeptide proved to be a potent tolerogenic molecule in vivo: One treatment of skin allograft recipient mice with (α1-α3)x2 was sufficient to significantly prolong graft survival, and four weekly treatments induced complete tolerance. Furthermore, (α1-α3)x2 was active as a soluble molecule and capable of inhibiting the proliferation of tumor cell lines, as does the full length HLA-G trimolecular complex. Thus, the synthetic (α1-α3)x2 polypeptide is a stable and simpler alternative to the full-length HLA-G molecule. It can be produced under GMP conditions, it functions as a soluble molecule, and it is at least as tolerogenic as HLA-G in vivo.

Keywords: engineered molecules; immune regulation; therapy; tolerance.

Figure 1.

(α₁-α₃)_x2 and (α₃-L)_x2 HLA-G synthetic polypeptides. Left: amino acid sequences of the HLA-G polypeptides used in this study. HLA-G α₃ polypeptides: linker sequences are underlined, and cysteines appear in bold type. HLA-G α₁-α₃polypeptide: sequence for HLA-G α₁ domain appears in italic type. Cysteines appear in bold type, including that in position 42 of the α1 domain, which mediates dimerization. Right: putative structures of the HLA-G polypeptide dimeric forms.

Figure 2.

HLA-G synthetic polypeptides binding to specific inhibitory receptors. Synthetic polypeptides were directly coated onto Bioplex beads, and recombinant B2M-HLA-G5 was captured by a specific antibody-coated beads. Positive signal was detected after specific binding of the inhibitory receptors.

Figure 3.

Tolerogenic function of HLA-G synthetic polypeptides in vivo. (α₁-α₃)_x2 increases skin allograft median graft survival time by 11 d with 1 injection and induces complete tolerance with a 4-injection treatment.

Figure 4.

Function of soluble HLA-G synthetic polypeptides in vitro. A) Different tumor cell lines were incubated in presence of (α₁-L)_x2, (α₃-L)_x2, and (α₁-α₃)_x2. B) A 36-h proliferation assay demonstrates that (α₁-α₃)_x2 strongly inhibits the proliferation of tumor cell lines.