The present invention concerns immune complexes for use in a diagnostic method, for use as a medicament and for use in a method of treatment.

Baio, G. et al., Mol. Imaging Biol. (2010) 12, pages 305 to 315 concerns a two- step in vivo tumor targeting by specific biotin-conjugated antibodies and ultrasmall superparamagnetic iron oxide (USPIO)-anti-biotin nanopartides as contrast agents for magnetic resonance imaging (MRI) at 1 .5 T (tesla). For demonstrating the functioning of the targeting human lymphoma cells expressing the CD70 surface antigen were injected either s.c. or i.v. to induce pseudo-metastases in NOD/SCID mice. Thirty micrograms of biotin-conjugated monoclonal anti-CD70 was injected i.v., followed 4 h later by 8 μιτιοΙ Fe/kg USPIO-anti-biotin. After 24 h, MRI was per- formed on T2 ^* and b-FFE sequences. Signal intensity (SI) was calculated before and after USPIO-anti-biotin administration. Subcutaneous xenografts showed a dishomogeneous 30% decrease in SI on T2 ^* with anti-CD70+USPIO-anti-biotin treatment. Pseudo-metastatic xenografts showed a slight reduction in SI on T2 ^* , but a 60% decrease in SI on b-FFE-weighted sequences. Prussian blue staining confirmed the presence of iron nanopartides in the excised tumors. This shows that MRI at 1 .5 T can detect tumors by a two-step in vivo biotin-based protocol, which may allow the targeting of any cell surface antigen. The document also discloses an in vitro MRI detection of cells labeled with biotin-labeled monoclonal anti-CD70 antibodies and USPIO-anti-biotin antibodies.

Goodwin, D. A. et al., Journal of Nuclear Medicine, vol. 39, no. 10, October 1998, pages 1813 to 1818 concern biological properties of biotin-chelate conjugates for pretargeted diagnosis and therapy with the avidin/biotin system. The document discloses the mixture of lndium-1 1 1 - or ⁸⁸ Y-labeled biotin-chelate conjugates with anti-IA ^k -MAb-streptavidin conjugates. The resulting complexes were given intravenously and the organ concentrations were measured at 3 hours. For this purpose, organs were sampled at 3 hours. The in vitro assay of streptavidin binding gave a lower than theoretical 4 : 1 ratio, based on the known streptavidin valence of 4. The measured biotin-chelate conjugates-to-streptavidin ratio was approximately 1 .87 : 1 ± 0.3. From Wang, Z. et al., Clinical Cancer Research, vol. 1 1 (19 suppl.), October 01 , 2005, pages 7171 s to 7177s monoclonal antibodies labeled with a trifunctional chelating reagent and biotin are known. The chelating reagent can chelate ligands in the form of radiometals, such as ¹¹¹ In. Such antibodies directed against specific antigens on tumor cells which antibodies comprised ¹¹¹ ln containing complexes were administered to rats. Subsequently, whole-body γ-camera imaging of the rats was done using a scintillation camera to determine the whole-body activity clearance.

WO 2009/012288 A2 relates to trifunctional imaging agents that include an anti- body for cell targeting, as well as a chelating moiety for sequestering radioisotopes and a fluorescing moiety for imaging. One of the imaging agents comprises a signaling agent in the form of the chelating moiety that is complexed with the radioisotope as a ligand and a targeting vector conjugated to the signaling agent. The targeting vector may be an antibody that binds with tumor cells. The document dis- closes a method for obtaining a diagnostic image of a mammal comprising administering to the mammal the imaging agent and exposing the mammal to an energy source, whereupon a diagnostic image of the mammal is obtained. The diagnostic image may be a positron emission tomography (PET) image. The problem to be solved by the present invention is to provide an alternative detection tool allowing an improved imaging of target cells or of a structure of a tissue in vivo. A further problem to be solved by the present invention is to provide a product for use as a medicament, a product for use in a method of treatment of a disease and a use of a product.

The problems are solved by the subject-matter of claims 1 , 1 1 , 12 and 15. Embodiments of the invention are subject-matter of claims 2 to 10, 13 and 14. According to the invention immune complexes are provided. The immune complexes are for use in a diagnostic method for detecting and localizing specific cells or a structure of a tissue in a human or animal body by an imaging technique in vivo. First antibodies are directed to an antigen located on the tissue or on the surface of said cells. The first antibodies are optionally labeled with first labels. The method comprises that the first antibodies and second antibodies directed to said first antibodies or to the first labels or the first antibodies and ligands specifically binding to the first labels are administered to said human or animal body, wherein said second antibodies or said ligands are labeled with second labels detectable by said imaging technique. The structure of said tissue or the cells is/are detected and localized in the human or animal body after administration of the first antibodies and second antibodies or of the first antibodies and the ligands by use of the imaging technique. The immune complexes of the invention are formed from the first antibodies and the second antibodies or from the first antibodies and the ligands ex vivo before administration of said immune complexes to the human or animal body.

Independent from each other the first antibodies and the second antibodies may be complete antibodies or fragments of antibodies, such as Fab or F(ab')2 fragments, which fragments have a specificity for the binding to a specific epitope in each case.

The inventors of the present invention recognized that the forming of the immune complexes outside the human or animal body allows - in contrast to the formation of immune complexes inside the human or animal body - a more intense and more defined labeling of the cells to be detected, i. e. a higher density of the second label on the cells. This is achieved by a ratio of the second antibodies to the first antibodies or of the ligands to the first antibodies in the immune complexes of at least 5 : 1 . Such a ratio can be obtained, e. g. if the second antibodies are polyclonal antibodies recognizing a plurality of different epitopes on the first antibodies or the first labels. Providing immune complexes according to the invention with such a ratio results in a multiplication of the number of second labels localized on each cell to which one of the first antibodies is bound. The forming of the immune complexes ex vivo enables a forming of the complexes under defined conditions with defined concentrations of the first and the second antibodies or of the first antibodies and the ligands thus allowing the formation of complexes with a relatively high number of second labels. The forming of the immune complexes ex vivo further allows to control the ratio of said second antibodies to said first antibodies or of said ligands to said first antibodies in the immune complexes by determining the amount of second labels per first antibody. If the first antibodies are labeled with first labels there is usually at least one first label per first antibody. Usually there is also at least one second label per second antibody or per ligand. In case of the use of ligands specifically binding to the first labels it is required to choose the amount of first labels according to the number of ligands that can be bound per la- bel such that at least 5 ligands can be bound per first antibody. For example, in case of avidin or streptavidin as first label it may be sufficient that two avidin or streptavidin molecules are bound per antibody because each avidin or streptavidin molecule can bind four biotin molecules as ligand. All of the first antibodies, the first labels, the second antibodies, the ligands and the second labels in each case may be identical. The first antibodies and/or the second antibodies in each case may be monoclonal or polyclonal antibodies.

In contrast to this the first antibodies are highly diluted when administered to the mice and only a part of the administered second USPIO-anti-biotin antibodies finds and binds to the first antibodies bound to the target cells in the method known from Baio et al.. This results in a low density of the USPIO-labels on the target cells and therewith to a low resolution when detecting the target cells in the animal body.

Even in case of the method described by Goodwin et al. the ratio of the biotin-che- late conjugates to streptavidin was only 1 .87 : 1 ± 0.3 though complexes were formed in vitro. This shows that care has to be taken to achieve the ratio of second antibodies to first antibodies or of ligands to first antibodies of at least 5 : 1 , e. g. by providing the second antibodies or the ligands in excess, giving the immune complexes enough time to form and/or by controlling the ratio by measurement of the amount of the second labels in relation to the amount of first antibodies before administration of the immune complexes to the human or animal body. The meas- urement of the amount of the second labels can be performed by measurement of a specific feature of the second labels, such as radioactivity.

The higher the density of second labels on the specific cell is, the better is the resolution that can be achieved with the imaging technique. The high density of sec- ond labels provided by the immune complexes according to the invention allows a very precise localization of the specific cells or tissue structure to which the immune complexes are bound in the human or animal body. The immune complexes of the invention provide a detection tool that allows a highly specific and sensitive imaging of target cells or of a structure of a tissue in vivo. These immune com- plexes can be used as a diagnostic tool particularly for the diagnostic detection of accumulations of few cells or a small tissue structure.

According to the present invention the imaging technique may be MRI and the second labels may be or may comprise paramagnetic iron oxide particles, in par- ticular superparamagnetic iron oxide (SPIO) particles, in particular particles comprising or consisting of FeO-Fe2O3. In case of paramagnetic iron oxide particles such as SPIO the above-mentioned measurement of a specific feature of the second labels for measurement of the amount of the second labels can be the measurement of iron concentration.

Alternatively, the imaging technique may be positron emission tomography (PET) and the second labels may be or may comprise positron-emitting radionuclides, in particular 2-deoxy-2-( ¹⁸ F)fluoro-D-glucose ( ¹⁸ F-FDG). Alternatively, the imaging technique may be scintigraphy and the second labels may be or may comprise ra- dionuclides. Any other imaging technique and corresponding labels may also be used. The antigen may be any marker for the tissue or for the structure of the tissue or any cell surface marker, in particular a marker for T cells, such as CD4 or CD8, a marker for B cells, such as CD19, or a marker for any other immune cells. The antigen may also be an immune cell differentiation marker, such as CD45RO or a mi- gration factor, i. e. a chemokine receptor, such as CCR6. CD4 is a characteristic surface antigen of T helper cells. CCR6 is a CC chemokine receptor protein present on the surface of immature dendritic cells, B cells and memory T cells.

The presence and/or the amount of the cells to be detected and localized in the human or animal body may indicate the presence and/or the stage of a disease to be diagnosed by the diagnostic method. The disease may be an inflammation, such as an inflammation caused by an autoimmune disease, e. g. in the gut of patients having a chronic inflammatory bowel disease or in the joints of patients having arthritis or rheumatism, in particular in the sacroiliac joints in patients having spondyloarthritis. A further disease that may be diagnosed by use of the immune complexes according to the invention may be an infection. In particular the immune complexes according to the invention allow the localization of the source of infection in patients having fever of unclear genesis. Furthermore, the disease to be diagnosed by use of the immune complexes according to the invention may be a cancer. In particular the immune complexes allow an early recognition of mi- crometastases, e. g. in case of a melanoma or a breast cancer, in particular mi- crometastases in local lymph nodes.

The cells to be detected and localized in the human or animal body may be iso- lated from the human or animal body prior to administration of the immune complexes according to the invention. After isolation, the cells may be labeled with the immune complexes according to the invention or with the first antibodies and the second antibodies or with the first antibodies and the ligands such that the immune complexes are formed on said cells. Then the cells can be administered to the hu- man or animal body together with the immune complexes bound to said cells. Alternatively or additionally the diagnostic method comprises that the immune complexes are administered orally or injected directly in the human or animal body, in particular subcutaneously, intramuscularly or intradermally or in a vein of the human or animal body. Alternatively or additionally the complexes may be in- jected directly into the peritoneum of the human or animal body or in a structure of the human or animal body, which structure shall be examined, such as an inflamed joint or tissue. "Injected directly" means that the immune complexes are not bound to a carrier such as previously isolated cells of the human or animal body. The cells may be any cells carrying said antigen as specific marker on the cell surface, in particular leucocytes, in particular chemokine receptor positive leucocytes, in particular CCR6 positive leucocytes.

Therapeutically active substances may be bound to the first antibodies and/or the second antibodies or to the first antibodies and/or the ligands. The therapeutically active substances may all be identical or may comprise different substances. The therapeutically active substances may be chemotherapeutic substances, in particular cytotoxic substances. In this way, the immune complexes according to the invention allow a specific treatment of the cells to be detected and localized and in particular the killing of these specific cells, e. g. inflammatory cells or in the case of cancer cells of the cancer, such as a cancer of blood.

The first labels can comprise or consist of biotin, phycoerythrin (PE), any other label or the therapeutically active substance. PE is useful when the diagnostic method is performed in the animal body. When the first labels comprise or consist of biotin the ligands may be avidin molecules or streptavidin molecules. Each avidin molecule or streptavidin molecule can bind four biotin molecules. When the first labels are formed by biotin it can be ensured that at least one avidin or streptavidin molecule is bound by each biotin molecule such that the amount of ligands can be defined very well. In case of avidin or streptavidin as first labels the amount of bound biotin molecules can vary. For example, Goodwin et al. disclose that in an in vitro assay the relation of biotin bound to streptavidin was approximately 1 .87 : 1 ± 0.3 though the theoretical ratio is 4 : 1 .

In one embodiment the first antibodies may be chimeric antibodies ,e. g., having a human Fab region and a murine Fc fragment as the first labels in each case. In this case the second antibodies may be a plurality of different human-anti-mouse Fc antibodies or polyclonal anti-mouse Fc antibodies labeled, e. g., with superparamagnetic iron oxide (SPIO) as second label. The invention also concerns immune complexes as specified above for use as a medicament, e. g. for the treatment of a disease caused by said cells, such as an inflammation, an infection or a cancer, in particular a cancer of blood. The disease may be a disease of a mammal, in particular a human being. The invention also concerns immune complexes as specified above for use in a method of treatment of a disease caused by said cells, such as an inflammation or a cancer, in particular a cancer of blood. The disease may be a disease of a mammal, in particular a human being. The treatment may be a targeted radionuclide therapy and the second labels may be radionuclides. Alternatively, the treatment may be a treatment by use of an alternating magnetic or an alternating electric field resulting in a heating of the second labels in said field wherein the second labels are paramagnetic/the paramagnetic iron oxide particles, in particular superparamagnetic/the supermagnetic iron oxide (SPIO) particles, in particular particles/the particles comprising or consisting of FeO-Fe2O3. In case of an alternating electric field the second labels are heated by electromagnetic induction. In case of an alternating magnetic field the second labels are forced into an oscillation movement resulting in a heating. The heating is then used for a heat therapy. By use of the heat therapy the specific cells or the tissue in which the specific cells are located can be specifically damaged or destroyed. The invention further concerns a use of the immune complexes as specified above in a non-therapeutic treatment in an animal model or in a non-diagnostic method for detecting and localizing specific cells or a structure of a tissue in an animal model.

The invention is further illustrated on basis of the following embodiments. shows a T2 ^* map before (left hand side) and after (right hand side) administration of the immune complexes. A legend correlating gray scale values to T2 ^* times is given on the outer right side of the figure.

Fig. 2 shows MRI images of a mouse at different echo times in an animal after injection.

Fig. 3a to 3d show histograms of T2 ^* times measured in the spleen of the animals before and after injection of the immune complexes according to the invention. Fig. 4a and 4b show prussian blue stainings of ironoxides in histological sections of the spleen of an animal treated with the immune complexes (SPIO-positive cells in the area within the black lines) (Fig. 4b) and of an animal not treated with the immune complexes (some physiologically iron-positive siderophages indi- cated by arrows) (Fig. 4a).

Immune complexes according to the invention were prepared by incubating 20 μΙ_ PE-labeled anti-CCR6-antibodies (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) with 40 μΙ_ anti-PE-antibodies labeled with FeO-Fe2O3 MicroBeads (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) in 160 μΙ_ MACS buffer (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) for 15 min at 4 °C. The spleen of two healthy C57BL/6 mice (animals 1 and 2) was imaged in vivo before and after administration of 100 μΙ of the immune complex via tail vain using MRI. All measurements were carried out on a 7T horizontal bore small animal system (Bruker Biospec, Bruker, Ettlingen, Germany) using a 35 mm quadrature bird- cage. A multi echo gradient echo sequence was used to acquire images at 8 different echo times (TE) (TE: 2.13 ms, 6.13 ms, 10.13 ms, 14.13 ms, 18.13 ms, 22.13 ms, 26.13 ms, 30.13 ms).

Resolution parameters were 20 slices, 0.75 mm slice thickness, no gap between slices, field of view 4 x 3 cm ² , matrix size 256 x 192. This results in a total volume of (4.0 x 3.0 x 1 .5) cm ³ and an in-plane resolution of 156 m x 156 μιτι. To minimize partial volume effects the imaging slices were orientated so that they are perpendicular to long axis of the spleen. From the obtained images T2 ^* times were determined for every voxel by fitting to the model a ^* exp(-TE/T2 ^* ). The spleen was manually selected in all slices. Afterwards the median of the values in the selected voxels was calculated.

The application of the immune complexes decreased the T2 ^* times in spleen as follows:

Table 1

Fig. 1 shows a T2 ^* map before (left hand side) and after (right hand side) administration of the immune complexes. A legend correlating gray scale values to T2 ^* times is given on the outer right side of the figure. Fig. 1 and the above table show a clear negative contrasting of the spleen tissue by shortening the T2 ^* relaxation times of the spleen due to the administration of the immune complexes. The spleen is indicated by a black circle.

Fig. 2 shows the first three echo images (TE: 2.13 ms, 6.13 ms, 10.13 ms, left to right) of a slice in the center of the spleen for animal 1 after administration of immune complexes. The signal of the spleen decays strongly between images, demonstrating the fast T2 ^* relaxation. The spleen is indicated by a black circle.

Images obtained with animal 2 are not shown. They were very similar to those ob- tained with animal 1 .

Fig. 3a to 3d show histograms of the measured relaxation times. The number of voxels is plotted against the relaxation times given on the x-axes. Fig. 3a shows the values obtained from the spleen tissue of animal 1 before administration of the immune complexes. The resulting median was 4.4 ms. Fig. 3b shows the values obtained from the spleen tissue of animal 1 after administration of the immune complexes. The resulting median was 3.0 ms. Fig. 3c shows the values obtained from the spleen tissue of animal 2 before administration of the immune complexes. The resulting median was 4.2 ms. Fig. 3d shows the values obtained from the spleen tissue of animal 2 after administration of the immune complexes. The resulting median was 2.9 ms.

Fig. 3a to 3d clearly show the shortening of the T2 ^* relaxation times in the spleen tissues after application of the immune complexes according to the invention.

Animals were sacrified 24 hours after injection of immune complexes. Fig. 4b shows a Prussian blue staining (black cells in the gray-scale picture) of a histological section of the spleen confirming the presence of iron nanoparticles in the spleen in the perifollicular regions (area between black lines in Fig. 4b).

Fig. 4a shows the Prussian blue staining of a histological section of a representative spleen of an animal without administration of immune complexes used as a negative control. Only singular spleen cells physiologically storing iron particles were found in the negative control indicated by arrows.