 Thomas Grüning MD MSc, Dept of Nuclear Medicine, Derriford Hospital, Plymouth PL6 8DH, United Kingdom  +44-1752-792280  Thomas.Gruning@phnt.swest.nhs.uk Biokinetics of 99m Tc-labelled antibodies in the rat − potential new lymphotropic tracers T. Grüning, G. Wunderlich, J. Kropp Department of Nuclear Medicine, University of Technology, Dresden, Germany Introduction Current status of lymph node imaging. Lymph nodes are involved in several pathologic entities, chiefly primary and secondary lymph node malignancies, autoimmune disease, non-autoimmune inflammations, and lymphoedema. Routine modalities for imaging lymph nodes include lymphoscintigraphy following subcutaneous injection of a radiolabelled colloid, X-ray lymphography following contrast injection into a cannu- lated lymphatic vessel and CT with intravenous contrast. MRI is not established in clinical routine due to the lack of suitable contrast media which are currently under development (SPIO). Aim of this study. We propose lymph node scintigraphy as a new modality where after intravenous injection of a radiolabelled targeting agent an image of all lymph nodes in the body can be obtained. This study aims to identify suitable targets and targeting agents. Target structures within lymph nodes comprise B and T lympho- cytes, dendritic cells (DC) and germinal centres (zones of high cell proliferation in the medulla). A good target structure for lymph node scintigraphy would be one with low expression at earlier stages of development of B and T cells, which mainly takes place in the bone marrow and thymus. Targets investigated in this study are: − CD23 is a low-affinity IgE receptor (FcεRII) found on mature B cells, follicular DC and activated macrophages. − CD38 is expressed on B cells within germinal centres, but also B and T precursors, plasma cells and activated T cells. − CD80 (B7.1) is found on B cells within germinal centres, and CD86 (B7.2) on activated B cells, DC and monocytes. Both are important for co-stimulation. − Immunoglobulin D is expressed on the surface (sIgD) of mature B cells. CD45 (LCA, B220) is expressed on all haematopoeitic cells and was used as a control. Materials and Methods Antibodies against rat antigens were obtained from Serotec: anti-CD23 (clone 2G8), anti-CD38 (90) and anti-IgD (MCA190), and Caltag: anti- CD45 (clone OX-1), anti-CD80 (3H5) and anti-CD86 (24F). Labelling. 99m Tc-labelling was performed using 2-iminothiolane (Traut’s reagent) as interchelating agent. 50 µg of antibody were incu- bated with 10 µl of 2-iminothiolane (Pierce) in PBS (10 mg/ml) for 1 hr. Non-bound iminothiolane was removed by ultrafiltration (Micro- con 30). 50 µl of a DTPA kit containing 7.5 µg SnCl 2 ⋅ 2 H 2 0 were added to the purified sample, followed by 100-200 µl of 99m Tc- generator eluate (100-1000 MBq) after 5 mins, and then incubated for 15 mins. The labelling yield was determined using ITLC (ITLC SG, Gelman) with 0.9 % NaCl. The labelled antibody was purified by ul- trafiltration, rinsed and centrifuged. Biokinetic studies. Ethics committee and local government approval was obtained. Eight-weeks old female Wistar rats were obtained from Charles River. Animals were anaesthetised and 50 MBq of radiolabelled antibody was injected into the footpad of one animal, and into the tail vein of another animal. Animals were placed prone on a plastic tray, and this was put directly on the collimator of a gamma camera (Siemens MultiSPECT III with LEHR collimators). Dynamic images were ac- quired up to 2 hrs p.i. using standard parameters for 99m Tc. Region-of- interest technique was used to determine the time-activity course in inguinal lymph nodes. A thin absorbent pad was placed under the lower abdomen of each animal to allow continuous urine collection without disturbing imaging. A blood sample was obtained 2 hrs p.i. Animals were sacrificed by CO 2 inhalation 24 hrs p.i., imaged and dissected. The radioactivity of most organs was measured in a well-counter (Cobra III, Packard), and that of high activity/high volume organs (liver, gut, residual body) was determined using the gamma camera, with an ap- propriate activity standard. Results Scintigraphic images at 2 hrs p.i. show uptake in lymph nodes with all antibodies investigated (fig. 1). The lymph node kinetics of 99m Tc- labelled antibodies after footpad injection is shown in fig. 2. Antibodies to co-stimulatory molecules CD80 and CD86 showed a maximum accumulation of about twice the initial value as well as good retention over time, whereas all other antibodies showed a steady decline of activity in the lymph node. Activity in serum was particularly low (table 1) and activity in the liver (fig. 3) particularly high with anti-CD80 and -CD86, perhaps suggesting a high degree of cell-bound activity. No lymph node uptake can be seen following i.v. injection (fig. 1). Table 1. Labelling yield, urinary excretion within 2 hrs p.i. (expressed as percentage of activity injected) and serum activity 2 hrs p.i. (expressed as counts/l/MBq injected), including protein- bound activity. sIgD CD23 CD38 CD80 CD86 CD45 labelling yield 42 % 68 % 54 % 87 % 72 % 22 % urinary excretion 37 % 24 % 27 % 34 % 28 % 47 % serum activity - i.v. injection - of which protein-bound - footpad injection 1400 58 % 730 1400 21 % 140 2000 21 % 210 980 42 % 110 910 40 % 84 300 52 % 100 sIgD CD23 CD38 CD80 CD86 CD45 Figure 1. Scintigraphic im- ages 2 hrs after i.v. (top) or footpad (bottom) injection. The footpad images show injection sites in one foreleg and one hindleg, kid- neys and bladder. 0% 50% 100% 150% 200% 250% 300% 0 30 60 90 120 mins relative activity (initial=100 %) sIgD CD23 CD38 CD80 CD86 CD45 0% 20% 40% 60% 80% 100% sIgD CD23 CD38 CD80 CD86 CD45 activity distribution 24 hrs p.i. residual body blood kidneys lung spleen liver Figure 2. Time-acitivity course in inguinal lymph nodes up to 2 hrs p.i. Figure 3. Activity distribution 24 hrs p.i. Activ- ity in organs not shown was included under 'residual body' and was as follows: heart, thyroid and thymus < 1 %, blood 1-4 %, stomach 2-4 %. Discussion When immunoglobulins are used as lymphoscintigraphic agents, spe- cific and lasting uptake in lymph nodes depends on the binding speci- ficities of the antibodies. Antibodies directed against co-stimulatory molecules CD80 and CD86 showed a high and persistent uptake in lymph nodes after footpad injection, but no lymph node uptake after i.v. injection. Further studies will seek to optimize delivery of suitable antibodies to lymph nodes following i.v. injection.