A Novel Allogeneic Cell Therapy That Targets Pathogenic Autoantibodies for the Treatment of Immune Thrombocytopenia
Authors: Patel-Hett Sunita , Giannini Silvia , Peters Christian , Dykstra Brad , Lehmann Marcus , Russo Ashley J , Kohnke Dora E , Carpenter Ryan , Tomczak Lindsay , Lazarus Douglas , Masuko Sayaka , Leung Amanda , Meredith Angelique , Pete Sophia , Lee Po-Shun , Falb Dean 
Published: November 2021

Abstract
Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts (&lt;100x10 9/L) and increased risk of bleeding. ITP is predominantly driven by immunoglobulin G (IgG)-autoantibodies directed against platelet surface antigens resulting in platelet destruction by the spleen and/or impaired platelet production by bone marrow megakaryocytes. Approximately 60% of ITP patients have measurable levels of anti-platelet autoantibodies in plasma and about 70% of them have autoantibodies directed against the fibrinogen receptor glycoprotein (GP) IIbIIIa.
Current treatment strategies for ITP include non-specific immunosuppression (steroids, rituximab), inhibition of platelet clearance (immunoglobulins, splenectomy, anti-D immunoglobulin, and the Syk inhibitor fostamatinib) and stimulation of platelet production (thrombopoietin receptor agonists). In addition, therapeutics targeting the neonatal Fc receptor, responsible for IgG recycling, are under clinical investigation for ITP. Despite these treatment options, some patients with ITP are refractory or have inadequate responses to existing therapy.
Here we describe a novel allogeneic cellular therapy, entitled platelet-like cells (PLC), that specifically targets pathogenic IgG for the treatment of thrombocytopenia in ITP. PLC are produced by differentiating a human induced pluripotent stem cell (hiPSC) line into megakaryocyte-like cells (MLC) which are further processed in a proprietary bioreactor that mimics the bone marrow environment and induces the release of anucleate PLC which are then cryopreserved. Analyses of PLC demonstrate sizes ranging between 65 nm and 10 μm. Expression of GPIIbIIIa protein as measured by enzyme-linked immunosorbent assay (ELISA) is evident in all particle sizes. PLC were administered intravenously (IV) into NOD-scid IL2Rgamma null (NSG) mice to evaluate their pharmacokinetics and biodistribution. PLC were rapidly cleared from the mouse circulation and predominantly distributed to the mouse liver where they colocalized primarily with Kupffer cells. PLC clearance through the liver was significantly inhibited by pre-treating mice with liposomes expressing phosphatidylserine (PS), indicating that one mechanism of PLC clearance is dependent on PS exposure on the PLC outer plasma membrane.
The ability of PLC to bind anti-GPIIbIIIa autoantibodies was evaluated in an in vitro assay where PLC were incubated with ITP patient plasmas and subsequently removed by centrifugation and filtration. The concentration of anti-GPIIbIIIa autoantibodies remaining in the plasmas decreased following PLC treatment (96.6% ± 2.4%).
The ability of PLC to clear anti-human GPIIbIIIa antibodies in circulation was evaluated by using a passive mouse model of ITP. Here, a fluorescently labeled mouse anti-human GPIIbIIIa monoclonal antibody (PAB-1) was dosed intravenously (IV) into NSG mice to achieve a circulating level of antibody comparable to those observed in ITP patients. The GPIIbIIIa antibody persisted in circulation for over 24 hours following a single dose in untreated mice and did not induce clearance of mouse platelets. Following administration of PLC in the passive ITP model, a decrease in antibody fluorescence in blood was observed within 2 minutes and full antibody clearance was observed at 3 hours post dosing. Furthermore, clearance of the antibody was observed to be both dose responsive and specific since PLC treatment did not affect the antibody concentration of an isotype matched control antibody. PLC activity in clearing the anti-GPIIbIIIa antibody was observed in all particle sizes. Fluorescence quantification of liver and spleen tissue demonstrated that PLC driven anti-GPIIbIIIa antibody clearance occurred preferentially in the liver. Taken together, these data indicate that by specifically binding and rapidly removing anti-platelet antibodies from circulation, PLC may disrupt the platelet degradation mechanisms underlying ITP pathogenesis and restore platelet counts.

Disclosures
Patel-Hett: PlateletBio: Current Employment. Giannini: Platelet Biogenesis: Current Employment. Peters: Platelet Biogenesis: Current Employment. Dykstra: Platelet Biogenesis: Current Employment. Lehmann: Platelet Biogenesis: Current Employment. Russo: Platelet Biogenesis: Current Employment. Kohnke: Platelet Biogenesis: Current Employment. Carpenter: Platelet Biogenesis: Current Employment. Tomczak: Platelet Biogenesis: Current Employment. Lazarus: Platelet Biogenesis: Consultancy. Masuko: Platelet Biogenesis: Current Employment. Leung: Platelet Biogenesis: Current Employment. Meredith: Platelet Biogenesis: Current Employment. Pete: Platelet Biogenesis: Current Employment. Lee: Platelet Biogenesis: Current Employment. Falb: Platelet Biogenesis: Current Employment.