Fig 1.
An autologous hinge as an Ab lock to enhance the selectivity and maintain the host immunity of Infliximab.
We used an autologous IgG1 hinge as Ab lock to cover the antigen-binding site of Infliximab by using MMP-2/9 substrate linker to generate pro-Infliximab. Upon protease activation at the RA region, the Ab lock was released, and the pro-Infliximab could specifically activate and neutralize TNFα at the disease site to inhibit RA progression. Ab, antibody; IgG1, immunoglobulin G1; MMP, matrix metalloproteinase; RA, rheumatoid arthritis; TNFα, tumor necrosis factor α.
Fig 2.
The Ab lock inhibits the binding ability and TNFα downstream signaling of Infliximab.
(A) The binding ability of Infliximab (○), pro-Infliximab (□), and MMP-2/9–activated pro-Infliximab (■) was assessed by TNFα-based ELISA. (B) TNFα-neutralizing ability of pro-Infliximab and Infliximab with or without MMP-2/9 through luciferase NF-κB reporter assay. TNFα treatment was used as a control for reporter activity. The values are mean ± SEM, and the asterisks indicate a significant difference, ****P < 0.0001. Error bar: standard error of triplicate determinations. Underlying data can be found in S1 Data. Ab, antibody; MMP, matrix metalloproteinase; NF-κB, nuclear factor kappa B; NS, no significance; TNFα, tumor necrosis factor α.
Fig 3.
The Ab lock can prevent the response of the anti-Id Ab to Infliximab.
(A) The anti-I-Id Ab–coated 96-well plate was incubated with different concentrations of Infliximab-biotin (○), pro-Infliximab-biotin (□), Infliximab-biotin preincubated with MMP-2/9 (●), or pro-Infliximab-biotin preincubated with MMP-2/9 (■), respectively, and then analyzed the neutralizing ability of anti-I-Id Ab by ELISA. (B) To further investigate the effect of anti-I-Id Abs on the restoration ability of TNFα binding of pro-Infliximab by protease. The Infliximab- or pro-Infliximab–coated plates were treated with different concentrations of anti-I-Id Abs. After removing unbound anti-I-Id Abs, we incubated the samples with or without MMP-2/9 and examined the TNFα-binding ability by TNFα-biotin and HRP-conjugated streptavidin. The values are mean ± SEM. Error bar: standard error of triplicate determinations. Underlying data can be found in S1 Data. Ab, antibody; anti-I-Id, anti-Infliximab idiotypic; HRP, horseradish peroxidase; MMP, matrix metalloproteinase; TNFα, tumor necrosis factor α.
Fig 4.
Evaluation of the PK properties and biodistribution of pro-Infliximab and Infliximab.
The DBA/1J mice were intraperitoneally injected with 131I-labeled Infliximab (○) or 131I-labeled pro-Infliximab (□). The blood was collected at different time and the radioactivity was detected via the γ counter to measure the (A) PK properties of 131I-labeled Infliximab and 131I-labeled pro-Infliximab. The biodistribution of 131I-labeled Infliximab and 131I-labeled pro-Infliximab in several tissues and organs in DBA/1J mice at 12 h (B), 24 h (C), 96 h (D), and 168 h (E) after injection. PK properties and biodistribution are expressed as radioactivity %ID/g. Error bar: standard error of triplicate determinations. Underlying data can be found in S1 Data. DBA/1J, dilute brown non agouti; %ID/g, percent injected dose per gram; PK, pharmacokinetic.
Table 1.
The Ab lock does not change the PK characteristics of Infliximab.
Fig 5.
The pro-Infliximab is activated at the disease site and suppresses disease development.
The hTNFα-transgenic 1006 mice were intraperitoneally injected with 50 μg Infliximab or pro-Infliximab. After 24, 48, 96, and 168 h, the blood and hind paw specimens were collected and using HRP-conjugated antihuman IgG Fab Ab for detecting the level of active and inactive pro-Infliximab in (A) serum and (B) joint tissue (paw) by western blot. The β-actin as internal control. I means Infliximab, pro-I means pro-Infliximab. The Tg197 mice (n = 8) were intraperitoneally injected with 10 mg/kg Infliximab (○), 10 mg/kg pro-Infliximab (■), or saline (◆). All mice were dosed twice weekly for 6 weeks. (C) Arthritic score of each group was monitored every week to measure the therapeutic efficacy. Representative HE stained sagittal tissue sections of the ankle joints in Tg197 mice after treatment with PBS (D), Infliximab (E, F), and pro-Infliximab (G) for 6 weeks. Representative histopathology images at 25× magnification. Bars = 1 mm. (H) The histopathology score of the paw in PBS, Infliximab, and pro-Infliximab–treated Tg197 mice. The values are mean ± SD, and the asterisks indicate a significant difference, *P < 0.01, **P < 0.001. Error bar: standard error of octuplicate determinations. Underlying data can be found in S1 Data. Ab, antibody; E, bone erosion; H, synovial hyperplasia; HE, hematoxylin–eosin; HRP, horseradish peroxidase; IgG; NS, no significance; P, pannus; T, tendonitis; Tg197 mice, hTNFα-transgenic mice; TNFα, tumor necrosis factor α.
Fig 6.
Comparison of the protective ability between pro-Infliximab and Infliximab on the host defense of TNFKO and Tg1278 TNFKO mice against Listeria infection.
The Tg1278 mice (n = 3) were intraperitoneally injected with 1 and 10 mg/kg of pro-Infliximab, Infliximab, or control saline and challenged with lethal dosage of L. monocytogenes (×104 CFU) 2 h later. The blood CFU counts (A) and representative HE staining (B) of liver tissues were tested to compare the host defense of Listeria infection. The arrows and circles represent the sites of infection identified as big well-shaped granuloma structures containing necrotic hepatocytes and inflammatory cells. Bars = 500 μm. (C and D) Survival rate after PBS, Infliximab. or pro-Infliximab treatment at low dose (1 mg/kg) (C) and high dose (10 mg/kg) (D) in L. monocytogenes-infected mice. The values are mean ± SEM and the asterisks indicate a significant difference, **P < 0.01, ***P < 0.001, ****P< 0.0001. Error bar: standard error of triplicate determinations. Underlying data can be found in S1 Data. CFU, colony-forming unit; HE, hematoxylin–eosin; NS, no significance; Tg1278 mice, hTNFα-transgenic mice; TNFα, tumor necrosis factor α; TNFKO, TNFα knockout.