New thrombopoietic growth factors

Abstract

Although development of first-generation thrombopoietic growth factors (recombinant human thrombopoietin [TPO] and pegylated recombinant human megakaryocyte growth and development factor [PEG-rHuMGDF]) was stopped due to development of antibodies to PEG-rHuMGDF, nonimmunogenic second-generation thrombopoietic growth factors with unique pharmacologic properties have been developed. TPO peptide mimetics contain TPO receptor-activating peptides inserted into complementarity-determining regions of Fab (Fab 59), attached to the IgG Fc region (AMG 531), or pegylated (Peg-TPOmp). Orally available, TPO nonpeptide mimetics (eltrombopag, AKR-501) bind and activate the TPO receptor by a mechanism different from TPO and may have an additive effect to TPO. TPO agonist antibodies are monoclonal antibodies activating the TPO receptor but modified in size [TPO minibodies; ie, VB22B sc(Fv)(2)] or immunoglobuln type (domain subclass-converted TPO agonist antibodies; ie, MA01G4G344). All second-generation thrombopoietic growth factors stimulate growth of TPO-dependent cell lines via JAK2/STAT signaling pathways and increase platelet counts in animals. When tested in healthy humans, TPO peptide and nonpeptide mimetics produced a dose-dependent rise in platelet count. AMG 531 and eltrombopag markedly increase platelet counts in patients with immune thrombocytopenic purpura, without significant adverse effects. One or more second-generation thrombopoietic growth factors should soon be clinically available for treating thrombocytopenic disorders.

Figure 1

Structure of rhTPO. Recombinant human TPO (rhTPO) is a fully glycosylated TPO made in Chinese hamster ovary (CHO) cells. It contains the TPO receptor binding domain (Mpl-binding domain) and the carbohydrate-rich COOH terminal domain. Red arrows indicate alpha helical areas; blue ovals denote areas of glycosylation. Illustration by Paulette Dennis.

Figure 2

Mechanism of activation of the TPO receptor by TPO. TPO binds the distal CRM (CRM 1) of the inactive TPO (c-Mpl) receptor and creates an activated receptor that initiates many downstream signal transduction events (see “First-generation thrombopoietic growth factors”). Although drawn as such here, the existence of preformed inactive dimers of the TPO receptor has not yet been convincingly demonstrated. Illustration by Paulette Dennis.

Figure 3

Structure of Fab 59. Fab 59 (A) is a fully human Fab containing one TPO agonist peptide (blue stripe) in the heavy-chain CDR3 and a second TPO agonist peptide in the light-chain CDR2, which activate the TPO receptor. In contrast, a related Fab, FabX4b (B), containing only one TPO agonist peptide, is not able to activate the TPO receptor until made dimeric by an anti-tag IgG antibody (C) that binds the hemagglutinin (HA) tag (purple ball) on FabX4c. Illustration by Paulette Dennis.

Figure 4

Mechanism of activation of TPO receptor by some TPO nonpeptide mimetics. TPO nonpeptide mimetics such as eltrombopag activate the TPO receptor by a mechanism different from TPO (see “TPO nonpeptide mimetics”). Although drawn here to suggest direct binding of eltrombopag to the TM region of the TPO receptor, eltrombopag may bind elsewhere on the TPO receptor but may have its effect mediated by unique structures in the TM region. Most TPO nonpeptide mimetics do not compete for binding with rhTPO and have a biologic effect additive to that of rhTPO. Illustration by Paulette Dennis.

Figure 5

Structure of eltrombopag. Eltrombopag [3′-{N′-[1-(3,4-Dimethyl-phenyl)-3-methyl-5-oxo-1,5-dihydropyrazol-4-ylidene]hydrazino}-2′-hydroxybiphenyl-3-carboxylic acid] has an acidic (COOH) group at one end, lipophilic (CH₃) groups at the other end, and a metal chelate group in the center, which create a potent, orally available TPO nonpeptide agonist. Illustration by Paulette Dennis.

Figure 6

Structure of VB22B sc(Fv)₂ minibody. A noncovalent (55 kDa) “diabody” (middle of figure) can be generated by taking the VH and VL regions of a whole anti-Mpl monoclonal IgG (left) and coupling them via a 5-amino acid linker (GGGGS). VB22B sc(Fv)₂ is made by binding one VH and one VL fragment together with a 15-amino acid linker [(GGGGS)₃] and then binding 2 of these constructs together with the same 15-amino acid linker (middle). Both the diabody and the VB22Bsc(Fv)₂ bind and activate the TPO receptor (right). Illustration by Paulette Dennis.

Figure 7

Structure of MA01G4G344 TPO agonist antibody. MA01G4G344 is a novel anti-Mpl agonist antibody that has an upper hinge region of human IgG3 and the other parts of human IgG4. The IgG3 upper hinge improved agonist activity of the whole IgG. The EC₅₀ value of MA01G4344 in the UT7/TPO proliferation assay was 0.01 to 0.1 nM, comparable to rhTPO. Illustration by Paulette Dennis.