Immunomodulatory activity of humanized anti-IL-7R monoclonal antibody RN168 in subjects with type 1 diabetes

Abstract

Background: The cytokine IL-7 is critical for T cell development and function. We performed a Phase Ib study in patients with type 1 diabetes (T1D) to evaluate how blockade of IL-7 would affect immune cells and relevant clinical responses.

Methods: Thirty-seven subjects with T1D received s.c. RN168, a monoclonal antibody that blocks the IL -7 receptor α (IL7Rα) in a dose-escalating study.

Results: Between 90% and 100% IL-7R occupancy and near-complete inhibition of pSTAT5 was observed at doses of RN168 1 mg/kg every other week (Q2wk) and greater. There was a significant decline in CD4+ and CD8+ effector and central memory T cells and CD4+ naive cells, but there were fewer effects on CD8+ naive T cells. The ratios of Tregs to CD4+ or CD8+ effector and central memory T cells versus baseline were increased. RNA sequencing analysis showed downmodulation of genes associated with activation, survival, and differentiation of T cells. Expression of the antiapoptotic protein Bcl-2 was reduced. The majority of treatment-emergent adverse events (TEAEs) were mild and not treatment related. Four subjects became anti-EBV IgG+ after RN168, and 2 had symptoms of active infection. The immunologic response to tetanus toxoid was preserved at doses of 1 and 3 mg/kg Q2wk but reduced at higher doses.

Conclusions: This trial shows that, at dosages of 1-3 mg/kg, RN168 selectively inhibits the survival and activity of memory T cells while preserving naive T cells and Tregs. These immunologic effects may serve to eliminate pathologic T cells in autoimmune diseases.

Trial registration: NCT02038764.

Funding: Pfizer Inc.

Keywords: Clinical Trials; Diabetes; Immunology; T cells.

Conflict of interest statement

Conflict of interest: KCH serves as scientific advisor to Provention Bio, Semma, Forkhead, and Bristol Myers Squibb. PAG serves as a consultant to Bristol Myers Squibb, Lilly, Kamada, Tolerion, and Viacyte and is cofounder and shareholder of IM Therapeutics. SEG served on advisory boards for Tolerion, Caladrius, and Immunomolecular Therapeutics and led a DSMB for Novo Nordisk. GW, TJ, CU, XW, SP, BG, and PDG are employees of Pfizer Inc. and may own stock/options in the company. ML, SLB, and MS were employees of Pfizer Inc. at the time of this research. JHP serves as a consultant for Insulet, Mannkind, Novo Nordisk, and Sanofi. JBM serves as a consultant for Boehringer Ingelheim, NovoNordisk, Bayer, and Aegerion and is a speaker for Aegerion, Dexcom, Janssen, and Mannkind. Grants to Washington University include Dexcom, AstraZeneca, Mannkind, and Novartis as sponsors.

Figures

Figure 1. Subject disposition.

PK, pharmacokinetics; PD, pharmacodynamics; SAE, serious adverse event.

Figure 2. IL-7 receptor (IL-7R) engagement by RN168 and phosphorylation of STAT5 in CD3+ T cells.

(A) IL-7R on CD3+ T cells. Baseline mean (±SD) values: placebo 1899.9 (565.6) (n = 7); RN168 1 mg 1327.7 (589.6) (n = 8); RN168 3 mg 1648.3 (376.2) (n = 9); RN168 6 mg 2245.8 (536.5) (n = 5); RN168 8 mg 2185.8 (722.8) (n = 8). (B) pSTAT5 in CD3+ T cells. Baseline mean (±SD) values: placebo 3750.4 (1393.9) (n = 7); RN168 1 mg 3681.7 (1665.7) (n = 8); RN168 3 mg 3707.7 (1321.4) (n = 9); RN168 6 mg 4066.0 (722.0) (n = 5); RN168 8 mg 3877.4 (1065.5) (n = 8).

Figure 3. Depletion of memory T cells with RN168 analyzed by flow cytometry.

(A) CD4+ naive T cells. Baseline mean (±SD) values: placebo 312.588 (127.118) (n = 7); RN168 1 mg 373.576 (139.967) (n = 8); RN168 3 mg 283.811 (146.604) (n = 9); RN168 6 mg 348.374 (135.402) (n= 5); RN168 8 mg 359.917 (165.903) (n = 8). (B) CD8+ naive T cells. Baseline mean (±SD) values: placebo 224.313 (142.442) (n = 7); RN168 1 mg 217.871 (96.265) (n = 8); RN168 3 mg 1 168.591 (119.241) (n = 9); RN168 6 mg 230.688 (42.754) (n = 5); RN168 8 mg 143.347 (73.942) (n = 8). (C) CD4+ effector memory T cells. Baseline mean (±SD) values: placebo 78.740 (37.003) (n = 7); RN168 1 mg 61.577 (24.059) (n = 8); RN168 3 mg 51.880 (24.289) (n = 9); RN168 6 mg 104.004 (28.278) (n = 5); RN168 8 mg 98.285 (57.377) (n = 8). (D) CD8+ effector memory. Baseline mean (±SD) values: placebo 69.043 (34.030) (n = 7); RN168 1 mg 101.506 (39.746) (n = 8); RN168 3 mg 56.524 (34.175) (n = 9); RN168 6 mg 107.370 (64.998) (n = 5); RN168 8 mg 113.887 (102.241) (n = 8). (E) CD4+ central memory T cells. Baseline mean (±SD) values: placebo 259.875 (57.937) (n = 7); RN168 1 mg 318.130 (161.006) (n = 8); RN168 3 mg 326.387 (138.693) (n = 9); RN168 6 mg 440.594 (171.652) (n = 5); RN168 8 mg 367.127 (154.881) (n = 8). (F) CD8+ central memory T cells. Baseline mean (±SD) values: placebo 52.228 (12.748) (n = 7); RN168 1 mg 44.133 (12.803) (n = 8); RN168 3 mg 60.531 (46.720) (n = 9); RN168 6 mg 78.612 (39.220) (n = 5); RN168 8 mg 53.712 (61.200) (n = 8).

Figure 4. Effects of RN168 on CD4+FoxP3+ Tregs assessed by flow cytometry.

(A) Tregs. Baseline mean (±SD) values: placebo 44.2733 (7.5852) (n = 7); RN168 1 mg 45.7443 (22.0758) (n = 8); RN168 3 mg 49.8629 (15.9373) (n = 9); RN168 6 mg 50.5640 (15.3193) (n = 5); RN168 8 mg 63.3043 (31.3837) (n = 8). (B) Ratio of Tregs to CD4+ effector memory T cells. Baseline mean (±SD) values: placebo 0.650 (0.248) (n = 7); RN168 1 mg 0.776 (0.348) (n = 8); RN168 3 mg 1.104 (0.483) (n = 9); RN168 6 mg 0.522 (0.216) (n = 5); RN168 8 mg 0.774 (0.377) (n = 8). (C) Ratio of Tregs to CD8+ effector memory T cells. Baseline mean (±SD) values: placebo 0.931 (0.830) (n = 7); RN168 1 mg 0.588 (0.304) (n = 8); RN168 3 mg 1.206 (0.824) (n = 9); RN168 6 mg 0.604 (0.321) (n = 5); RN168 8 mg 0.767 (0.655) (n = 8).

Figure 5. Gene expression changes in peripheral blood CD8+ and CD4+ T cells between day 85 and baseline in response to dosing with RN168.

(A) Heatmap of gene expression change of the top 50 differential protein-coding genes in CD4+ T cells. (B) Changes in expression of select immune-related genes (CCR6, RANK, Tbet, and GATA3) in CD4+ T cells. (C) Heatmap of change in expression of top 50 differentially expressed protein-coding genes in CD8+ T cells. (D) Changes in expression of select immune-related genes (LTK, CCR4, CD40LG, and KLRB1) in CD8+ T cells.

Figure 6. Dose-response analysis of RN168 average concentration (Cavg) and gene expression change between day 85 and baseline in CD8+ T cells.

(A) Correlation of select individual genes. (B) Association of IL-2RG. (C) Association of IL-7R expression.

Figure 7. Percentage change from baseline in Bcl-2 expression in CD3+ T cells.

(A) CD4+Bcl-2+ T cells. Baseline mean (±SD) values: placebo 3231.750 (982.634); RN168 1 mg 5973.00 (0); RN168 3 mg 3501.000 (507.441); RN168 6 mg 3178.000 (702.489); RN168 8 mg 3130.875 (1026.502). (B) CD8+Bcl-2+ T cells. Bcl-2 values in the RN168 1 mg/kg Q2wk group were unavailable after day 8 due to an interruption in assay reagent availability. Baseline mean (SD) values: Placebo 3399.750 (1332.569); RN168 1 mg 3218.000(0); RN168 3 mg 3434.500 (300.684); RN168 6 mg 3217.000 (657.476); RN168 8 mg 3255.000 (1557.702). Placebo, n = 7; RN168 1 mg/kgQ2wk, n = 8; RN168 3 mg/kgQ2wk, n = 9; RN168 6 mg/kg Q1wk, n = 5; RN168 8 mg/kg Q2wk, n = 8.

Figure 8. Effects of RN168 on clinical responses.

Each line represents an individual participant. The bold, colored lines represent the mean ± SD of the dosing cohort. (A) C-peptide AUC levels. (B) Tetanus toxoid challenge, by treatment group. The number of participants in each dosing regimen are: placebo, n = 7; 1 mg/kg Q2wk, n = 8; 3 mg/kd Q2wk, n = 9; 8 mg/kg, n = 8; 6 mg/kg QW, n = 5.