Development and preclinical characterization of AMG 329: a human antibody neutralizing FLT3 ligand

Abstract

The feline McDonough sarcoma-like tyrosine kinase 3 (FLT3)/FLT3 ligand (FLT3L) signaling pathway regulates the development and activity of dendritic cells (DCs) and other myeloid cells, including monocytes. FLT3L, DCs, and monocytes have been implicated in several autoimmune diseases. Here, we describe the development and characterization of a human immunoglobulin G1λ monoclonal antibody (AMG 329; formerly MEDI1116/VIB-1116/HZN-1116) targeting human FLT3L. AMG 329 was derived from a large human combined antibody display library; it was optimized to enhance affinity for FLT3L and reduce antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity. Binding affinity was determined by surface plasmon resonance interaction analysis. Specificity of FLT3L was measured using cell-based flow cytometry and an in vitro functional neutralization assay. ADCC activity was measured using an in vitro cell culture system. Toxicity and toxicokinetics were evaluated in cynomolgus monkeys during AMG 329 dosing (5-100 mg/kg; ≤ 27 weeks) and recovery (≤32 weeks). The AMG 329 antigen-binding region selectively bound to human and cynomolgus monkey FLT3L with affinities of 170 and 63 pM, respectively. AMG 329 specifically bound to and neutralized soluble and cell-bound human FLT3L and did not induce ADCC. AMG 329 administration generally reduced circulating plasmacytoid, conventional DC, and classical monocyte relative proportions in cynomolgus monkeys in a non-dose-dependent manner. Disruption of the FLT3/FLT3L signaling pathway presents a new potential therapeutic approach to treat autoimmune and inflammatory diseases. AMG 329 is a selective human monoclonal antibody antagonist of FLT3L that is currently being investigated in clinical studies.

Keywords: AMG 329; FLT3; FLT3 ligand; dendritic cells; monocytes; plasmacytoid dendritic cells.

Figure 1.

Cross-reactivity of 5D9 for human, cynomolgus monkey, and mouse FLT3L. 5D9 demonstrated binding to both human and cynomolgus monkey FLT3L but not mouse FLT3L. 5D9, parental antibody for AMG 329; Ab, antibody; FLT3-Fc, FLT3-Fc chimera protein; FLT3L, feline McDonough sarcoma-like tyrosine kinase 3 receptor ligand; IgG, immunoglobulin G; MFI, mean fluorescence intensity.

Figure 2.

AMG 329 neutralizes soluble and cell-bound human FLT3L. (a) Increasing concentrations of anti-FLT3L antibodies were preincubated with 96 pM human soluble FLT3L before being added to human FLT3-expressing RS4;11 cell cultures. FLT3 expression was quantified using a fluorescently labeled anti-FLT3 antibody and measured by flow cytometry. Increased fluorescence indicates a higher level of FLT3 on the cell surface, demonstrating neutralization of FLT3L activity. Each point represents the average of duplicate wells. (b) AM40/AMG 329 neutralizes the activity of cell-surface FLT3L on CD4+ T cells at concentrations greater than 1 nM. Increased FLT3 expression indicates a higher level of FLT3 on the cell surface, demonstrating the neutralization of FLT3L activity. Neutralization of cell-bound FLT3L was measured using FLT3L-expressing CD4+ T cells co-cultured with FLT3-expressing RS4;11 cells. The isotype control curve is at 0% expression. 5D9, parental antibody for AMG 329; CD, cluster of differentiation; FLT3, feline McDonough sarcoma-like tyrosine kinase 3 receptor; FLT3-Fc, FLT3-Fc chimera protein (positive control); FLT3L, FLT3 ligand; MFI, mean fluorescence intensity; pAb, polyclonal antibody.

Figure 3.

AMG 329 does not show antibody-dependent cellular cytotoxicity. IL7–treated CD4⁺ T cells and allogeneic PBMCs were incubated with anti-CD4, AMG 329, or hIgG-TM control. CD4⁺ T cells were treated with IL7 to upregulate FLT3L expression. CD4⁺ T cells were incubated with PBMCs at a 1:25 ratio and assayed with a range of antibody concentrations (anti-CD4, AMG 329, or hIgG-TM control) for 6 hours. Cytotoxicity was measured using a colorimetric assay to detect lactate dehydrogenase. Data are presented from one donor representative of six independent experiments of nonrepeating PBMC donors. Each point represents the average of triplicate wells. Error bars represent standard deviation. CD, cluster of differentiation; FLT3L, feline McDonough sarcoma-like tyrosine kinase 3 receptor ligand; hIgG, human immunoglobulin G; PBMC, peripheral blood mononuclear cells; TM, triple mutation.

Figure 4.

AMG 329 did not bind human C1q in vitro. Binding of AMG 329 to C1q was evaluated by ELISA. Serial dilutions of AMG 329, rituximab (human IgG1; positive control), and IgG4 S241P L248E (Abzena; hinge stabilized and Fc silenced; negative control) were incubated with purified human C1q. Anti-C1q-HRP was used to measure binding with the TMB microwell peroxidase assay. All samples were tested in duplicate and ran on two separate occasions. EC50, half maximal effective concentration; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; NA, not applicable; OD, optical density.

Figure 5.

Peripheral blood immunophenotyping evaluations in cynomolgus monkeys: pDcs over time. Data were pooled for males and females and presented as the percent of baseline of relative proportions (percent of CD45^brightSSC^dim lymphocytes and CD45^moderateSSC^moderate monocytes) during (a) the 5-week, 5-dose toxicity study and (b) the 27-week, 14-dose chronic toxicity study. *adjusted p < .05; **adjusted p < .01; ***adjusted p < .001 vs control. IQR, interquartile range; IV, intravenous; pDC, plasmacytoid dendritic cell; SC, subcutaneous.

Figure 6.

Peripheral blood immunophenotyping evaluations in cynomolgus monkeys: cDC1 counts over time. Data were pooled for males and females and presented as the percent of baseline of relative proportions (percent of CD45^brightSSC^dim lymphocytes and CD45^moderateSSC^moderate monocytes) during (a) the 5-week, 5-dose toxicity study and (b) the 27-week, 14-dose chronic toxicity study. *adjusted p < .05; **adjusted p < .01 vs control. cDC1, conventional dendritic cell 1; IV, intravenous; SC, subcutaneous.

Figure 7.

Peripheral blood immunophenotyping evaluations in cynomolgus monkeys: classical monocyte counts over time. Data were pooled for males and females and presented as the percent of baseline of relative proportions (percent of CD45^brightSSC^dim lymphocytes and CD45^moderateSSC^moderate monocytes) during (a) the 5-week, 5-dose toxicity study and (b) the 27-week, 14-dose chronic toxicity study. *adjusted p < .05; **adjusted p < .01 vs control. IV, intravenous; SC, subcutaneous.