Targeting Long-Lived Plasma Cells Is Essential For Treating Hemophilia A Inhibitors

The most critical and challenging problem for therapeutic treatment in hemophilia A patients is due to the formation of Inhibitory antibodies. It is hypothesized that long-lived plasma cells (LLPCs) play an important role in the persistent production of anti-FVIII antibodies in hemophilia A (HemA) inhibitor patients. The migration of plasma cells to the BM, where they become the LLPCs, is largely controlled by an interaction between the C-X-C type chemokine ligand 12 (CXCL12) produced by bone marrow (BM) stromal cells and its receptor CXC receptor4 (CXCR4; CD184) on plasma cells surface. Our previous data showed that administration of anti-murine CD20 (IgG2a) alone can deplete B cells significantly and reduce anti-FVIII inhibitor titers transiently in FVIII plasmid-treated HemA mice with pre-existing inhibitors, however, complete tolerance to FVIII was not achieved probably due to the persistence of LLPCs. We sought novel therapeutic strategies that target CXCL12/CXCR4 pathway to reduce/eliminate LLPCs and achieve the goal for long-term tolerance to FVIII in the HemA inhibitor mice. AMD3100, the CXCR4 antagonist, plus G-CSF inhibit the interaction of CXCL12 and CXCR4, thus facilitating the mobilization of stem cells and blocking the homing and retention of LLPCs. The combined treatment strategy used anti-CD20, G-CSF and AMD3100 to target B cells plus with the specific IL-2/IL-2mAb (JES6-1) complexes to target T cells for preventing both T and B cell-dependent anti-FVIII immune responses. Two groups of FVIII-primed inhibitor mice were treated with different combined immunomodulation regimens: (1) IL-2 complexes+AMD3100+G-CSF+anti-CD20, (2) AMD3100+G-CSF+anti-CD20. Control mouse groups were treated with each of the single regimens and FVIII only, or untreated as the naive control. All the treatments were administered one cycle per two weeks for 6 weeks and the therapeutic effects (FVIII activities) as well as immune responses (anti-FVIII inhibitors) were evaluated at different time points after treatment. Significant expansion of Treg cells reaching a 5∼7-fold increase on the peak days (day 3-7 after treatment) was observed in the IL-2/IL-2mAb complexes treated groups, whereas ∼95% of B cell populations were depleted in the anti-CD20 treated groups. In addition, administration of AMD3100 plus G-CSF significantly reduced circulating and bone marrow CXCR4⁺ plasma cells by flow cytometry analysis as well as decreased FVIII-specific plasma cells in bone marrow by ELISPOT assay. Except for the control groups, the two mouse groups treated with combined immunosuppressive regimens showed a significant reduction of inhibitory titers following the treatment. Long-term responses are being followed and second challenge with FVIII plasmid will be used to evaluate the induction of long term tolerance to FVIII. These combination regimens are highly promising in modulating/eliminating pre-existing anti-FVIII antibodies and inducing long-term tolerance in FVIII primed subjects.