Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α

Abstract

We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

Figure 1

Stability of golimumab in solution. Intact antibodies (A) and Fab fragments (B) were dialyzed, degassed and analyzed by differential scanning calorimetry with dialysate in the reference cell. Buffer-buffer scans run independently were used for data baseline subtraction. (C) golimumab formulated at 100 mg/mL and stored at the indicated temperatures was analyzed by size exclusion chromatography (SEC) at each time point to determine the % monomeric golimumab.

Figure 2

Golimumab neutralization of soluble TNFα and tmTNFα compared with other TNFα antagonists. Neutralization of cell cytotoxicity was compared using serial dilutions of golimumab (solid circles), infliximab (solid triangles), etanercept (open circles), adalimumab (open triangles) or negative control mAb (open squares) pre-incubated with 0.1 ng/mL of soluble TNFα (A) or murine K2 cells expressing human tmTNFα (B), followed by overnight incubation with KYM target cells. Each data point represents the mean of duplicate wells, and the error bars represent the range of the duplicate values. (C) Serial dilutions of the same proteins listed above were pre-incubated with 1 ng/mL of soluble TNFα followed by incubation for 4 hours on human umbilical vein endothelial cells. Iodinated anti-E-selectin antibody was used to detect expression of E-selectin on the cell surface. The data points represent the mean of duplicate wells and the error bars show the range.

Figure 3

Contribution of golimumab and infliximab to lysis of LPS-stimulated human PBMCs. Human PBMCs treated +/−1 µg/mL of LPS for 2 hours (A) or murine K2 cells expressing tmTNFα (B) were incubated with biotinylated golimumab (shaded area, no LPS; dark line, LPS-stimulated) or negative control mAb (light gray line, LPS-stimulated) and detected with streptavidin-phycoerythrin. PBMCs were gated on CD14⁺ monocytes and human IgG (100 µg/mL) was added to block FcγRs. Cell lysis using LPS-stimulated human monocytes (C) or murine K2 cells expressing tmTNFα (D) was determined following the addition of the indicated concentration of golimumab (solid circles), infliximab (solid triangles), or negative control antibody (open squares) followed by the addition of human PBMC as the source of immune effector cells. The extent of target cell lysis was quantitated after 2 hours. The data shown are the mean ±SEM of replicate wells (n = 2–6).

Figure 4

Golimumab and infliximab suppress disease activity in the human TNFα transgenic mouse model of arthritis. The change in arthritic index was monitored weekly following a single intraperitoneal injection at week 0 of 1 mg/kg (A) or 10 mg/kg (B) of golimumab (solid circles) and infliximab (solid triangles). Results for the vehicle control group (open squares) are shown on both graphs. Each data point is the mean (n = 10) ±SEM. The symbols indicate p < 0.05 compared with vehicle (*) or golimumab compared to the same dose of infliximab (#).

Figure 5

Golimumab preserves joint architecture. Representative images of the tibia-talus joint stained with hematoxylin and eosin (A–C) or Toluidine Blue (bottom, D–F) are shown from Tg197 transgenic mice treated with a single s.c. injection of PBS (A and D) or 30 mg/kg golimumab (B and E) alongside an untreated F1 non-transgenic mouse (C and F). All images are 20x magnification. (G) Hematoxylin and eosin-stained tissue sections from transgenic mice treated with PBS or with golimumab doses ranging from 1 to 30 mg/kg were ranked in order of severity (highest score representing the worse disease), then the ranks were grouped by treatment. The golimumab 30-mg/kg group was significantly different from the PBS group (p < 0.01). (H) The tibia-talus joints were scored for the extent of cartilage matrix degradation in mice treated with PBS (n = 4) or with golimumab doses of 1 mg/kg (n = 4), 3 mg/kg (n = 4), 10 mg/kg (n = 4), 30 mg/kg (n = 7). The 30-mg/kg group achieved a statistically significant reduction in cartilage damage compared with the PBS-untreated animals (p = 0.025). Overall severity and cartilage destruction are shown as mean ± SD, with arrows indicating articular cartilage.

Figure 6

Golimumab reduces serum level of proinflammatory cytokines and chemokines. Samples collected 23 d posttreatment were analyzed for granulocyte-macrophage colony-stimulating factor (GM-CSF; A), interleukin-6 (IL-6; B), granulocyte colony-stimulating factor (G-CSF; C), keratinocyte chemoattractant (KC; D), and interferon-inducible protein-10 (IP-10; E). Treatment groups included TNF transgenic mice treated with PBS or golimumab and non-transgenic wild type mice treated with PBS. The plotted values represent the mean +/− SEM.