Patients and study protocol

The study was approved by the local ethics committee. Patients fulfilled the criteria of rheumatoid arthritis (according to the American College of Rheumatology Criteria) and were treated with infliximab (3 mg/kg) with 8-week intervals at inclusion. Two female responders and two female non-responders to infliximab (age 24–71 years; duration of the disease 6–38 years) were included. Patients received at least five prior infliximab infusions. The European League Against Rheumatism response criteria were used to determine whether a patient was a responder or a non-responder.⁶ The presence of HACAs was not known at the start of the study. Any comedication was allowed; all included patients were treated with methotrexate.

Eight weeks after the last infusion of infliximab, patients were infused with 10 mg 740 MBq technetium-99m infliximab in 50 ml NaCl (0.9%) during 2 h. Simultaneously, they received, via the same infusion needle, 3 mg/kg (−10 mg) unlabelled infliximab in 250 ml NaCl (0.9%) in 2 h. A micropore filter (0.21 μm) was used in the infusion application system. Blood samples were withdrawn for further analysis directly before infusion (t = 0 h), during infusion (t = 5, 10 and 30 min; 1 and 2 h) and post infusion (t = 18 and 24 h). Images were recorded directly after infusion and 24 h later.

Imaging

Infliximab was coupled to ^99mTc as described previously.^7,899mTc-infliximab was purified on a PD10 column (PD10 column filled with Sephadex™ G25M, Amershon, Piscataway, New Jersey, USA) with 0.9% NaCl as an eluent and filter sterilised (endotoxin levels <5 units/ml). The radiochemical purity, using high-performance liquid chromatography and thin-layer chromatography, was >99.5%. The immunoreactivity of ^99mTc-infliximab, determined using preformed sepharose beads coated with TNF via a non-competing antibody to TNF,⁹ was >92%.

Whole-body images (matrix 1024×256) and spot views of joints (matrix 256×256) were recorded with an ECAM gamma camera (Siemens, Siemens Mediscle Techniek, Zoetermeer, The Netherlands). Regions of interest were drawn on digital images of the whole-body and on several organs. Whole-body counts were corrected for machine drift, physical decay and counting time differences. Geometrical means were used for analysis. A region of interest in the left ventricle of the heart, corrected for lung background, was used as measure for blood pool activity. Spleen uptake was also corrected for background activity. Ratios of organ uptake to blood pool activity of ^99mTc-infliximab were calculated.

Serum analysis

Infliximab was quantified as described⁹ with one modification. Infliximab binding was assessed by incubation with biotinylated rabbit immunoglobulin (Ig) G directed to the infliximab idiotype. Detection limit of this assay is about 0.1 μg infliximab/ml serum. Antibodies to infliximab were measured using a method described previously.¹⁰ The size of ^99mTc-infliximab was analysed by sedimentation analysis on sucrose gradients as described previously.¹¹

Administration of ^99mTc-infliximab to patients

Infliximab could be safely administered to the two responders (A and B). One non-responder (C) developed a light transient pressure on the chest, but the infusion could safely be continued. A serious infusion reaction was observed in the other non-responder (D), who developed a shock at 45 min of infusion. The infusion was immediately discontinued, followed by proper treatment. The severity of the infusion reaction did not allow this patient to continue the study. Blood samples were collected for up to 30 min; no image data were collected.

Imaging

Up to 24 h post infusion, ^99mTc-infliximab was mainly present in the blood pool (fig 1⇓). At 24 h, 100% and 98% of infused ^99mTc-infliximab were still present in the whole-body of responders A and B, respectively. A relatively faster plasma and whole-body clearance was found in the non-responder C; the whole-body retention at 24 h was 76%. Besides blood pool, liver, spleen and kidneys were also visualised. At 2 h, liver uptake in the non-responder was higher than that in the responders, with liver to blood pool ratios of 2.6, 1.5 and 2.2, respectively. Spleen to blood pool ratios showed similar differences. At 24 h, no significant differences in liver and spleen uptake were observed. Uptake of ^99mTc-infliximab was seen in clinically inflamed joints (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

 Whole-body images. Anterior and posterior whole-body images of a responder directly after (2 h) and 24 h post infusion.

Infliximab and anti-infliximab levels

Infliximab levels of responder A, negative for anti-infliximab, rose to about 45 μg/ml on infusion (fig 2A⇓). Responder B had low levels of anti-infliximab (82 AU/ml) at t = 0. During infusion, anti-infliximab disappeared and infliximab levels rose to 35 μg/ml (fig 2B⇓). High anti-infliximab levels of 1008 and 1641 U/ml were found in both non-responders, C and D respectively (figs 2C, D⇓). The highest level was detected in patient D, who developed the infusion reaction (fig 2D⇓). In both non-responders, all infused infliximab was bound to circulating anti-infliximab within the first 30 min, resulting in low infliximab levels. At 30–60 min of infusion, the concentration of infliximab overruled that of anti-infliximab, resulting in increasing infliximab concentration (fig 2C⇓).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

 Infliximab and anti-infliximab levels in the sera of responders A and B (A,B) and two non-responders C and D (C,D). (D) The x and y axes were adjusted because of lacking samples after 30 min of patient D and the lower amount of infliximab in the samples up to 30 min.

Infliximab–anti-infliximab complexes

Samples containing sufficient radioactivity (⩾30 min) were analysed. Sucrose gradients of serum of responder A showed a sharp peak of ^99mTc-infliximab at fraction 18, exactly the position at which free IgG was found in the nephelometer (fig 3A⇓). This implies absence of infliximab–anti-infliximab complexes. In the responder B, a minor radioactivity shift was seen at 30 and 60 min, probably reflecting the formation of small amounts of small complexes that disappeared later (fig 3B⇓). Higher amounts of small complexes were found in the non-responder C at 30 and 60 min, with a peak at fraction 15 (fig 3C⇓). Their sedimentation properties corresponded to the formation of 1:1 complexes between infliximab and an IgG antibody to infliximab. The serum of non-responder D showed complexes of various sizes (at fraction 15 and lower fractions), including very large ones (molecular weight >1 000 000; fig 3D⇓). From 1 h onwards, no complexes were found in any of the patients.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

 Sucrose gradients of sera of responders A and B (A,B) and of non-responders C and D (C,D). The measured radioactivity in the fractions as a percentage of the maximum radioactivity at each time point (% of maximum) is shown. The values on the y axis in D have been adjusted to the lower amounts of radioactivity in the 30-min sample to visualise the size of the antibody complexes in this patient.