A new method for radiolabeling of human immunoglobulin-G and its biological evaluation.

BACKGROUND: Radiolabeled human Immunoglobulin-G (hIgG) has demonstrated its utility in inflammation and infection imaging. However, the present method of radiolabeling hIgG is time-consuming and complex.
OBJECTIVE: To develop a simplified method of radiolabeling hIgG with technetium-99m ((99m)Tc) via a nicotinyl hydrazine derivative ((99m)Tc-HYNIC-hIgG) and its biological evaluation.
RESULTS: In vitro and in vivo studies showed that (99m)Tc-hIgG prepared by this method was fairly stable in physiological saline and human serum till 24 h. Only 4.3% degradation of the radiolabeled drug was seen till 24 h. Blood clearance pattern of the radiopharmaceutical exhibited biphasic exponential pattern. Biodistribution of (99m)Tc-HYNIC-hIgG in mice was observed up to 24 h. Significant accumulation of the radiotracer was found in liver (4.93 %), kidney (3.67%) and intestine (2.12 %) at 4 h interval by 24 h interval, it was reduced to 1.99%, 2.18% and 1.93 % respectively. Significant amount of radioactivity in liver, kidney and intestine suggest hepatobilliary as well as renal route of clearance for (99m)Tc-HYNIC-hIgG. The anterior whole body and spot scintigraphy images showed increased uptake of 99mTc-HYNIC-hIgG, with the area seen as a focal hot spot, indicating good localization of the radiolabeled hIgG at the site of infection.
CONCLUSION: The present findings indicate that (99m)Tc-HYNIC-hIgG holds great potential for the scintigraphy localization of inflammation. The shelf life of the developed kit, when stored at (-) 20°C was found to be at least 3 months.

The conventional radiological imaging techniques (computed tomography, ultrasonography and nuclear magnetic resonance imaging) are useful in the diagnosis of anatomical abnormalities. Generally, nuclear medicine imaging techniques are more useful in picking up the inflammatory and infectious foci, which are the early stage processes of the late anatomical manifestations. Immunoscintigraphy with radiolabeled antimicrobial antibodies is an approach to detect infection, and radionuclide technetium-99m (99mTc) is extensively used in radio diagnostics in nuclear medicine.[1] Tc-99m has been used in the last few decades to label many kits. Its nuclear features of 6-hour half-life (t½) and gamma energy of 140 KeV make it one of the best radionuclides for imaging purposes; with a t½ long enough to permit acquisition of complete study without any unnecessary dose of radiation to the patient. Among the variety of radiopharmaceuticals for early detection of infection and inflammation, 67Ga, 99mTc-nanocolloids and 111In- or 99mTc-labeled leukocytes have particularly been used frequently. However, due to non-specificity, high radiation dose and poor imaging characteristics, the first two agents are not preferred these days.[23] Radiolabeled leukocytes although offers good specificity, but its labeling procedure is tedious, time consuming and requires skilled manipulation.[4]

111Indium-labelled-Human Immunoglobulin-G (111In-hIgG) has been demonstrated to be a useful radiopharmaceutical for imaging focal sites of inflammation in both animal and human models.[4-6] The mechanism of 111In-hIgG localization was originally postulated to involve binding of Fc-portion of the hIgG to specific receptor on inflammatory cells. Micro-autography studies however have revealed that this is not the case though.[7] In addition, it was recently demonstrated that although digestion of hIgG with endoglycosidase-F significantly reduced Fc-receptor binding, inflammation imaging was however not altered.[8] These observations suggest that inflammation localization is probably mediated by less specific mechanisms. It appears that nonspecific portion leak into expended protein space of inflammatory lesions play an important role in the localization of 111In-hIgG.[9] Since, diffusion of hIgG into this expended protein space occurs rapidly, early lesion detection should be possible if high count density images are acquired. Unfortunately, the limited photon flux of the usually administrated doses of 111In-IgG makes it difficult to image the early stage of this process due to long acquisition time required for recording a sufficient count density. Ever since then, there has been a tremendous interest to label hIgG with 99mTc and evaluating it for imaging such lesions.

In the present study, we report a novel method of radiolabeling hIgG with 99mTc using 6-Hydrazinopyridine-3-carboxylic acid (HYNIC) and tricine. HYNIC constitutes one of the most attractive functional coupling agents (CAs) to prepare Tc- 99m labeled radiopharmaceuticals for visualization of tumors, infection and thrombosis at early post-injection period.[5] HYNIC has been shown to act as a monodentate or bidentate ligand, forming a mixed ligand- 99mTc complex in presence of an appropriate co-ligand such as tricine.

Materials and Methods

Reagents and chemicals

Human immunoglobulin-G (hIgG) was commercially obtained from Sandoz Inc., USA. Tricine, 2-mercaptoethanol, HYNIC were purchased from Sigma-Aldrich, USA, and stannous sulfate was purchased from Merck. 99mTechnetium pertechnetate was obtained from BRIT (INMAS) India. Remaining reagents were of reagent grade.

Purification and conjugation of hIgG with HYNIC

Five hundred milligram of hIgG was dissolved in 10 ml distilled water and this solution was drawn onto slide-A-Lyzer dialysis cell and dialyzed overnight at 4°C using normal saline. The contents of dialysis cell were drawn through Millipore filter (0.22 micron) and 0.8 mg of HYNIC solution was added in 100 μl DMSO. The reaction mixture was incubated at room temperature in dark for 30 min. The pH was adjusted to 6.4 with 1.5 M sodium phosphate buffer and dialyzed for overnight at 4°C in a sterile dialysis cell using 1.5 M sodium phosphate buffer. Aliquots were made containing equivalent of 2 mg of hIgG. Finally, aliquots were dispensed into 15 ml vials, frozen overnight at -20°C, lyophilized, sealed under vacuum and stored in a refrigerator until use.

Tricine kit formulation for labeling hIgG-HYNIC with 99mTc

Hundred milligram of Tricine was dissolved in 8 ml of distilled water and acidified with 0.5 ml of 2.0 M HCl. The contents were made inert using gentle stream of Nitrogen gas. 0.5 ml of stannous sulphate solution (20 mg/ml) was added and pH adjusted to 5.5 using 2 M sodium hydroxide solution. The contents were filtered through 0.22 μ millipore filter and stored overnight at -20°C, lyophilized sealed under vacuum and stored in a refrigerator until use.

Radiolabeling of hIgG-HYNIC with 99mTc

99mTc-HYNIC-hIgG was synthesized according to procedures described previously,[1011] with slight modification. In brief, vial containing hIgG-HYNIC was thawed at room temperature and 60 μl of freshly prepared Trycine kit was added to it followed by addition of 20-30 mCi 99mTc-perthecnetate. The contents were dissolved by swirling the vial gently and incubated at room temperature for 20 min to complete the labeling reaction.

Quality control

The labeled product was subjected to ascending instant thin layer chromatography (ITLC). Strips (1´12 cm) of ITLC-SG, (Gelman, USA) were used as stationary phase and acetone was used as the mobile phase to separate free pertechnetate and 99mTc-HYNIC-hIgG, which moved with the solvent. Citrate buffer (pH 5.5) was used as the mobile phase to separate 99mTc-HYNIC-hIgG, and 99mTc-HYNIC.

Stability of 99mTc-HYNIC-hIgG

Stability studies of the radiolabeled antibody were performed as described previously.[12] One hundred microliters of the radiolabeled antibody was incubated in 1.9 ml of saline or human serum at 37°C. Small aliquots were withdrawn at different time intervals up to 24 h and radiolabeling efficiency was evaluated by ITLC using citrate buffer as mobile phase.

Trans-chelation study (Cysteine/DTPA challenge)

Aliquots of 0.5 ml of 99mTc-HYNIC-hIgG (100 μl) were challenged over a range of cysteine and DTPA concentrations (25–100 mmol/L) at 37°C for 1 h. The percentage of 99mTc displaced by cysteine and DTPA was determined by ITLC-SG using normal saline as mobile phase. (Labeled-HYNIC-HYNIC, Rf = 0.0–0.15, free 99mTc, Rf = 0.75–1.0 and Cystine/DTPA- 99mTc, Rf = 0.6–0.7).

Lipophilicity study

Hundred microliters of the radiolabeled antibody was mixed with 2.0 ml of dichloromethane and 1.9 ml of normal saline. The mixture was kept at room temperature for 1 h and radioactive counts of the two separated phases were taken using γ–counter (Capintec, USA) to determine per cent lipophilicity of the radiolabeled product.

Protein binding studies

One hundred microliters of 99mTc-HYNIC-hIgG, was mixed with 1.9 ml of fresh serum and the contents were kept at 37°C for 1 h. 2 ml of 10% Trichloric acetic acid was added to the mixture and the contents were centrifuged for 5 min at 3500 rpm. Per cent radioactive counts in supernatant and precipitate fractions were noted using g-counter (Capintec, USA) to determine extent of protein binding.

Biodistribution studies

All animal experiments were conducted in accordance with our institutional guidelines, and the experimental procedure was approved by the Institutional Animal Ethical Committee duly constituted for the purpose. Biodistribution experiments were performed by intravenous administration of 100 μl of 99mTc- HYNIC-hIgG (1 μCi) to strain-A mice (25–40 g; n = 10). Animals were sacrificed by decapitation at 4 h and 24 h after injection. Organs of interest, namely, heart, liver, lungs, spleen, kidneys, stomach, intestine and muscle were removed, washed with saline and weighed, and respective radioactivity counts were determined with the help of a g-counter (Capintec, USA). Data were expressed as percentage of administered radioactivity per g of organ.

Blood clearance study

200 μl of 99mTc-HYNIC-hIgG (100 μCi) was intravenously administered to rabbits (n = 5) through the ear vein. Blood samples were withdrawn from the other ear vein at various time intervals. Radioactivity per unit volume was calculated by counting a known volume from the sample at each time point. Whole blood was considered as 7% of the body weight to calculate the percentage of administered radioactivity present in total body blood.

Preparation of animal model and immunoscintigraphy

A group of three male New Zealand rabbits, weighing 2–3 kg were used in the immunogenicity studies. They were maintained on a normal diet. Infection was induced by inoculation of E. coli (ATCC 22923) into the left thigh muscle. After 4–5 days, when gross swelling was apparent in the infected thigh, 3 mCi of 99mTc-HYNIC-hIgG was injected intravenously via ear vein. The anesthetized animal was placed in the supine position and scanned under the gamma camera.

Anterior whole body image and spot views of the lower extremities were obtained hourly for the first 2 h and subsequently at 24 h. Acute aseptic inflammation was induced in three other New Zealand white rabbits, weighing 2–3 kg with turpentine. Commercial grade turpentine liquid was sterilized by ultrafiltration using a 0.45 nm bio-filter. Each animal received 0.1 ml of the sterile turpentine liquid by intramuscular injection into the right thigh muscle. Acute inflammatory lesion was developed on second days. The rabbits were then intravenously administered with 3.0 mCi of 99mTc-HYNIC-IgG via ear vein. The anesthetized animal was placed in the supine position and scintigraphy was done under the gamma camera.

Human imaging

Normal biodistribution study was carried out using 99mTc- HYNIC-hIgG scintigraphy in human volunteers. Briefly, 10.0 mCi 99mTc-HYNIC-hIgG (equivalent of 2.0 mg hIgG) was administered in normal male volunteers (n = 2) intravenously for the acquisition of imaging data. Anterior and posterior whole-body images were obtained immediately at 6 h and 24 h post-injection using a dual head Hawkeye gamma camera system (GE, Milwaukee, USA) using the inbuilt software Entegra Version-2. Regions of interest (ROI) were constructed at each time point for the whole body and all organs visualized. The geometric mean of activity in these regions was calculated and corrected for radioactive decay. Organ radioactivity, as a fraction of the administered dose, was calculated by using the immediate whole body as 100% of the injected dose. From the imaging results, the residence time for 99mTc-HYNIC-hIgG in the major source organs (heart chambers, kidneys, liver, lungs, spleen, and the remainder whole body) was calculated.

Statistical analysis

Data were expressed as mean of 6 experiments. Results of biodistribution experiments were statistically analyzed using a one-way ANOVA followed by the Dunnett post-hoc test. Differences were considered statistically significant when P values were less than 0.05.

Results and Discussion

The potential value of radiolabeled hIgG as an imaging agent for detecting focal infection has been previously reported.[13] An indirect 111 In-labelling via chelating group attached to the antibody has been exploited successfully;[14] this labeling approach is not preferred due to the variable binding affinity of 111In to the antibody's amino groups,[15] which may partly be located in the hypervariable region of the antibody molecule, thereby altering its immunoreactivity. The radiolabeling procedure being reported here for labeling hIgG with Tc-99m using HYNIC gives consistently high (>96%) radiolabeling efficiency with respect to the radiolabeled product [Table 1]. Stability of hIgG kit (shelf life) was also evaluated by storing the kit for different time periods. Results indicate that the kit is stable up to three months with only 1.6% reduction in radiochemical purity of the Tc-99m labeled product [Table 2]. In vitro stability study of radiolabeled hIgG was studied in saline at room temperature and in human serum at 37°C till 24 h [Table 3], Up to 4 h, no free pertechnetate was detected irrespective of the storage condition, indicating no leaching of Tc-99m from the product. Even at 24 h the free pertechnetate content were only 5.1% and 9.5% in case of serum and saline, respectively. High binding affinity of 99mTc-hIgG was ascertained by allowing the tagged protein to incubate with DTPA and cysteine at different molar ratios [Table 4]. DTPA at a molar concentration of 25 and 50 times that of hIgG decreased labeled hIgG by only 3.1% and 7.2% respectively. However, when DTPA was increased 100 times, the labeled percentage of hIgG was reduced by 11.83%. In case of transchelation with cysteine the reduction of the labeled hIgG was more pronounced. Even at a molar ratio of 25 times the decrease was about 5.8%, which increased to 23.7% at 100 times cysteine concentration. The above observations suggested a high binding affinity of hIgG–SH group for Tc-99m. Lipophilicity study of the labeled product showed good lipophilic character of the molecule [Table 5].

Table 1

Optimization of radiolabeling efficiency of 99mTc-hIgG with different concentration of reducing agent using ITLC method of quality control

Table 2

Stability of hIgG kit after storing these kits for different time period

Table 3

Stability of 99mTc-hIgG when incubated at room temperature in saline and at 37°C in human serum, respectively, using ITLC method of quality control

Table 4

Different molar concentration of DTPA and cysteine were mixed with 99mTc-hIgG. Labeling efficiency was measured by ITLC. Each value is the mean of three separate experiments of the same preparation

Table 5

Lipophilicity profile of the 99mTc-HYNIC-hIgG

Biodistribution of 99mTc-HYNIC-hIgG in mice at 4 h and 24 h after intravenous administration is shown in Figure 1. Among the various organs studied, significant accumulation of the radiotracer was found in liver (4.9%), kidney (3.7%) and intestine (2.1%) at 4 h interval. By 24 h interval, it reduces to 2.0%, 2.2% and 1.93% respectively. The remaining organs (heart, lungs, spleen, stomach and muscles) had very low accumulation of the radiotracer. Low activity in the stomach, suggest an insignificant amount of free pertechnetate in the 99mTc-hIgG preparation, further confirming the in vivo stability of the radiolabeled hIgG molecule. Significant amount of radioactivity in liver, intestine and kidneys suggest hepatobiliary as well as renal route of clearance for 99mTc-HYNIC-hIgG. Blood clearance data in rabbits after intravenously administering 2-3 mCi of 99mTc-HYNIC-hIgG exhibited slow and biphasic clearance [Figure 2]. At 1 h, 15.1% of the injected radioactivity was present in blood, which reduced to 4.93% by 24 h.

Figure 1

Biodistribution of 99mTc-HYNIC-hIgG. The animal were intravenously administered with 1 uCi tracer and the radioactivity in various organ was measured at 4 and 24 h. Each value is the mean of 6 mice expressed in % dose administered per organ

Figure 2

Blood clearance of 99mTc-HYNIC-hIgG in rabbit after administering it intravenously. Each value is the mean of three independent experiments

Anatomical delineation of the extent of focal inflammation is critical to clinical management of infectious processes, both for diagnosis and for monitoring the response to therapy. In our experiments, acute inflammatory lesion was developed within 3 days following the injection of sterile turpentine liquid. An opaque creamy whitish lesion developed at the site of injection surrounded by inflammatory tissue. No pus was found in the inflamed tissue during autopsy and the entire inflamed thigh muscle tissue measured about 3 cm in size. The site of inflammation appeared as an area of diffuse uptake of 99mTc- HYNIC-hIgG during scintigraphy. In animals that were injected with E. coli to generate an infection model, abscess formation occurred within 7–8 days post inoculation of E. coli. A small firm mass measuring less than 1 cm in size was found in the infected thigh with noticeable gross swelling. The anterior whole body and spot scintigraphy images at 2 h and 24 h showed increased uptake of 99mTc-HYNIC-hIgG, with the area seen as a focal hot spot, indicating good localization of the radiolabeled hIgG at the site of infection [Figure 3]. The image of an inflammatory lesion of patient [Figure 4] obtained using a SPECT camera after administering 99mTc-HYNIC-hIgG indicates not only the quality of the kit and radiopharmaceutical preparation, but also clinical usefulness.

Figure 3

(a) 2 h and (b) 24 h anterior and posterior view showing 99mTc-hIgG accumulation in rabbit. Right thigh shows the acute infection developed by sterile turpentine while right thigh of rabbit shows the site of abscess formation by E. coli

Figure 4

99mTc-HYNIC-hIgG distribution in human volunteer at 24 h

The present method of radiolabeling hIgG with HYNIC is a simple, easy to perform radiolabeling procedure that gives high radiolabeling yield with minimum colloid formation. Our experimental observations indicate good accumulation of 99mTc-HYNIC-hIgG at the infection and/ or inflammation site. 99mTc-HYNIC-hIgG imaging data indicated higher lesion to background ratio in the infected foci as compared to the inflammatory foci The technique may therefore have a good diagnostic utility in the differentiation of infection from inflammation in clinical situations.