Purification of Bioactive Natural Antibodies to PD-1 Known as Regulatory Rheumatoid Factor from Human Blood Plasma

Background/Objectives: Regulatory rheumatoid factor (regRF) is a recently discovered population of natural anti-idiotypic antibodies carrying a shared paratope specific to the PD-1 molecule. Normally, regRF exerts a cytotoxic effect on PD-1-expressing T lymphocytes, including autoreactive ones, thereby controlling their expansion. The specificity for PD-1 indentifies regRF as a promising therapeutic agent for suppressing autoimmune reactions via the PD-1 pathway. The aim of this study was to develop gentle methods for regRF isolation from healthy blood plasma, which would preserve the bioactivity of regRF. Methods: Ammonium sulfate precipitation was used for crude regRF purification. A search for suitable affinity sorbents was carried out. The purified regRF was tested for its bioactivity. Results: Ammonium sulfate precipitation at 35% saturation, followed by extraction with 28% saturated ammonium sulfate solution, allowed the separation of regRF from a portion of non-target IgG and high-molecular-weight fibrinogen. RegRF showed high affinity for Cibacron Blue and no binding to Protein A. Sequential affinity chromatography on immobilized Cibacron Blue and Protein A enabled the purification of regRF from non-target immunoglobulins and low-molecular-weight fibrinogen. The purified regRF samples showed binding to PD-1 and reduced the PD-1+CD4+ T lymphocyte count in vitro. Conclusions: Thus, we have identified sorbents and conditions for the purification of biologically active regRF.