WO2010097182A1 - Procédé d'imagerie in vivo de la lymphangiogenèse de nœuds lymphoïdes par tomographie par émission d'immuno-positrons et marqueurs pour celle-ci - Google Patents

Procédé d'imagerie in vivo de la lymphangiogenèse de nœuds lymphoïdes par tomographie par émission d'immuno-positrons et marqueurs pour celle-ci Download PDF

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WO2010097182A1
WO2010097182A1 PCT/EP2010/001044 EP2010001044W WO2010097182A1 WO 2010097182 A1 WO2010097182 A1 WO 2010097182A1 EP 2010001044 W EP2010001044 W EP 2010001044W WO 2010097182 A1 WO2010097182 A1 WO 2010097182A1
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antibody
lymphatic
labeled
lyve
lymph nodes
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Michael Detmar
Viviane Mumprecht
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Eidgenoessische Technische Hochschule Zurich ETHZ
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/06—Devices, other than using radiation, for detecting or locating foreign bodies ; Determining position of diagnostic devices within or on the body of the patient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/41—Detecting, measuring or recording for evaluating the immune or lymphatic systems
    • A61B5/414—Evaluating particular organs or parts of the immune or lymphatic systems
    • A61B5/415—Evaluating particular organs or parts of the immune or lymphatic systems the glands, e.g. tonsils, adenoids or thymus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/41—Detecting, measuring or recording for evaluating the immune or lymphatic systems
    • A61B5/414—Evaluating particular organs or parts of the immune or lymphatic systems
    • A61B5/418—Evaluating particular organs or parts of the immune or lymphatic systems lymph vessels, ducts or nodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B6/00—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment
    • A61B6/02—Arrangements for diagnosis sequentially in different planes; Stereoscopic radiation diagnosis
    • A61B6/03—Computed tomography [CT]
    • A61B6/037—Emission tomography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/48—NMR imaging systems
    • G01R33/54—Signal processing systems, e.g. using pulse sequences ; Generation or control of pulse sequences; Operator console
    • G01R33/56—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution
    • G01R33/5601—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution involving use of a contrast agent for contrast manipulation, e.g. a paramagnetic, super-paramagnetic, ferromagnetic or hyperpolarised contrast agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B6/00—Apparatus or devices for radiation diagnosis; Apparatus or devices for radiation diagnosis combined with radiation therapy equipment
    • A61B6/02—Arrangements for diagnosis sequentially in different planes; Stereoscopic radiation diagnosis
    • A61B6/03—Computed tomography [CT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/08—Clinical applications
    • A61B8/0833—Clinical applications involving detecting or locating foreign bodies or organic structures

Definitions

  • the invention relates to methods for in vivo imaging of the lymphatic system such as lymph nodes, e.g. for detection and/or monitoring of lymphangiogenesis, using Immuno- Positron Emission Tomography and other imaging methods, to markers for these imaging methods and to a procedure for obtaining such marker systems.
  • Metastatic spread is a characteristic trait of most tumors types and is the cause for the majority of cancer deaths.
  • metastasis to the regional lymphatic system i.e. in particular the lymph nodes
  • metastasis to the regional lymphatic system is the first step of tumor dissemination and a prognostic indicator for the progression of the disease.
  • the presence of tumor cells in the lymph nodes is a major determinant for the clinical management of cancer patients.
  • regional lymph nodes or in cases of breast cancer and melanoma patients only the tumor draining (sentinel) lymph nodes, are invasively analyzed, i.e. are dissected and sections are analyzed for metastases.
  • this procedure is elaborate and associated with significant morbidity and costs and raises the demand for a sensitive, non-invasive, and simpler method to detect metastasis to the lymph nodes.
  • lymphatic vasculature lymphatic vasculature
  • primary cancers were shown to induce lymphangiogenesis within the sentinel lymph nodes even before the on-set of metastasis. Once the metastases arrived in the lymph nodes, lymphangiogenesis was further enhanced and promoted metastasis to distant lymph nodes and organs.
  • Expanded lymphatic networks were also found in the metastatic lymph nodes of human melanoma patients and in metastatic lymph nodes of human breast cancer patients, correlating with distant metastasis. These studies show that lymph node lymphangiogenesis is a significant process in humans.
  • vascular endothelial growth factor VEGF-A and VEGF-C vascular endothelial growth factor VEGF-A and VEGF-C, amongst others, that are produced by tumor and tumor-associated stromal ceils. These factors are drained into the lymph nodes or are produced in there once the cancer cells have arrived.
  • Lymph node lymphangiogenesis can be used as a prognostic indicator to screen for cancer metastasis. Therefore the aim of this invention is to establish methods to image this process non-invasively in vivo using methods such as positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence-mediated-tomography (FMT), magnetic resonance imaging (MRI), ultrasound, bioluminescence imaging, and the like.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • FMT fluorescence-mediated-tomography
  • MRI magnetic resonance imaging
  • ultrasound bioluminescence imaging
  • bioluminescence imaging and the like.
  • the non-invasive in vivo imaging is carried out using labelled antibodies (or optionally also labeled non-antibody binders) to lymphatic specific epitopes. Labelled means in this context that the labels attached to and/or integrated into the antibodies are able to facilitate and/or make possible imaging using any of the above- mentioned methods.
  • Radiolabelling in this context means that a system is attached or integrated into the antibody which emits positrons.
  • PET imaging visualizes the distribution of a positron (e + ) emitting radiotracer administered to an animal or a person in three- dimensions at very high sensitivity. Emitted positrons move through the tissue until they have lost enough energy to react with a tissue electron. The photons resulting from the annihilation process of positrons and electrons are then detected and used for image reconstruction.
  • positron e +
  • SPECT Single photon emission computed tomography
  • Fluorescence-mediated-tomography if the antibody is coupled to an applicable fluorophor for FMT.
  • Magnetic resonance imaging if the antibody is coupled to a suitable MRI-contrast agent.
  • Ultrasound if the antibody is linked to a suitable ultrasound active contrast agent such as microbubbles.
  • Bioluminescence imaging if the antibody is coupled to a suitable molecule able to produce bioluminescence.
  • the object of the present invention is therefore a method for non-invasive in vivo imaging of the lymphatic system, comprising the steps of delivery of at least one labeled antibody to or labeled non-antibody binder to at least one non-intracellular, surface accessible protein present in or expressed on lymphatic vessels, or in the extracellular matrix of lymphatic vessels, to a patient; allowing the at least one labeled antibody or labeled non-antibody binder to distribute and eventually accumulate in the lymphatic system (it is noted that during this time span further and continued delivery of the labelled antibody or labelled non-antibody binder is possible); acquiring at least one image associated with the label of the labeled antibody or labeled non-antibody binder of the lymphatic system.
  • Preferentially labeled antibody or labeled non-antibody binder should be selected such that it does not inhibit lymphangiogenesis to avoid interference with the object of observation.
  • a system is chosen as the protein for the antibody or non-antibody binder which has a redundant function.
  • imaging or data acquisition for image generation can be carried out on a single (two- dimensional or three-dimensional) picture basis but also the time development can be analysed, be it under continuing delivery of the labelled antibody or labelled non-antibody binder or after a single shot delivery of such a system.
  • the expression antibody shall be meant to include (for example IgG but also IgM) antibodies to image lymph node lymphangiogenesis, other antibody isotypes or formats, e.g. small immuno proteins, single chain variable fragment antibodies (scFv), diabodies or minbodies etc.
  • the delivery of the of at least one labeled antibody or labeled non-antibody binder is carried out by, subcutaneous, , intravenous, and/or intraperitoneal injection, preferably by intravenous injection. Typically delivery is carried out by intravenous injection of the labeled antibody or labeled non- antibody binder.
  • the at least one labeled antibody to or labeled non-antibody binder can be labeled with a radiolabel and the image can be acquired using positron emission tomography.
  • the at least one labeled antibody or labeled non-antibody binder can be labeled with a radionuclide applicable for single photon emission computed tomography and the image can be acquired using single photon emission computed tomography.
  • single photon emission computed tomography SPECT
  • SPECT single photon emission computed tomography
  • the at least one labeled antibody to or labeled non-antibody binder can be labeled with a fluorophor applicable for fluorescence-mediated-tomography and the image can be acquired using fluorescence-mediated-tomography.
  • fluorescence- mediated-tomography FMT
  • FMT fluorescence- mediated-tomography
  • the at least one labeled antibody or labeled non- antibody binder can be labeled with a magnetic resonance imaging contrast agent and the image can be acquired using magnetic resonance imaging.
  • magnetic resonance imaging MRI
  • the antibody is coupled to a suitable MRI-contrast agent.
  • the at least one labeled antibody or labeled non- antibody binder can be labeled with a ultrasound imaging contrast agent and the image can be acquired using ultrasound imaging. So ultrasound imaging can be used, if the antibody is linked to a suitable system such as a microbubble.
  • the at least one labeled antibody or labeled non- antibody binder can be labeled with a system able to produce bioluminescence and the image can be acquired using bioluminescence imaging. So bioluminescence imaging can be used, if the antibody is coupled to a suitable molecule able to produce bioluminescence.
  • the at least one labeled antibody or labeled non-antibody binder to a protein is one to a protein expressed on the cell surface membrane by lymphatic endothelial cells or which is enriched in the extracellular matrix, and which preferably is essentially not or only weakly expressed on B or T cells or tissues adjacent to the lymph nodes.
  • the step of allowing the at least one labeled antibody or labeled non-antibody binder to distribute and eventually accumulate in the lymphatic system is carried out for at least 1 h, preferably for at least 2 or at least 3 h, even more preferably for at least 12 h.
  • the step of acquiring at least one image associated with the label of the labeled antibody or labeled non-antibody binder of the lymphatic system is carried out in a time window of 1-6Oh, preferably in a time window of 15-50 h, after initiation of delivery of at least one labeled antibody to or labeled non-antibody binder to at least one non-intracellular, surface accessible protein expressed on lymphatic vessels, or secreted into the extracellular matrix by lymphatic endothelial cells to a patient.
  • the at least one labeled antibody can be a 18 F, 45 Ti, 52 Fe, 55 Co, 61 Cu, 62 Zn, 64 Cu, 68 Ga, 71 As,
  • the non-intracellular, surface accessible protein expressed on lymphatic vessels, or secreted into the extracellular matrix by lymphatic endothelial cells can be preferentially selected from the following group:
  • LYVE-I Activin A receptor, type 1 (ACVRl), Collagen type IV Al (COL4A1), Collagen type IV A2 (COL4A2), EPH receptor Bl (EPHBl), Insulin-like growth factor 1 receptor,
  • MMP- 13 Matrix metalloproteinase 13
  • Neogenin Neogenin
  • PAU Semaphorin 3A
  • SEMA 3A Semaphorin 3A
  • TMC Tenascin-C
  • lymphatic endothelial cells in vitro or in vivo, which can be used as targets to image lymph node lymphangiogenesis are as follows: Aquaporin-1 (Gannon, B.J., and CJ. Carati. 2003. Lymphatic research and biology 1 :55-
  • DC-SIGN CD209 (Martens, J.H., et al. 2006. The Journal of pathology 208:574-589),
  • Endoglin (Clasper, S., D. et al. 2008 ;Hirakawa, S., et al, 2003), Endothelial-specific-molecule-l ESM-I (Shin, J.W., et al. 2008. Blood 1 12:2318-2326.),
  • Ephrin B4 receptor (Makinen, T., et al. 2005. Genes & development 19:397-410),
  • ESAM Endothelial cell adhesion molecule
  • Fibroblast growth factor receptor-3 (Shin, J.W., et al. 2006. MoI Biol Cell 17:576-584),
  • Hepatocyte growth factor receptor (c-met) (Kajiya, K., et al. 2005. Embo J 24:2885-2895),
  • Integrin alpha 1 (Hong, Y.K., et al. 2004, Faseb J 18 :1111-1113), Integrin alpha 2 (Hong, Y.K., et al. 2004, Faseb J 18 :11 11-1113),
  • Integrin alpha 4 beta 1 (Garmy-Susini, B., et al. 2007. Methods in enzymology 426 :415-
  • Integrin alpha 9 (Huang, X.Z., et al. 2000. Molecular and cellular biology 20:5208-5215),
  • ICM-I Intracellular adhesion molecule 1
  • Jagged-1 Hirakawa S., et al. 2003. The American journal of pathology 162:575-586
  • LA 102 (Ezaki, T., et al. 2006. Anatomy and Embryology 211 :379-393),
  • Macrophage mannose receptor (Takahashi, K., et al. 1998. Cell and tissue research
  • MF AP3 Microfibrillar associated protein 3 (MF AP3) (Hirakawa S., et al. 2003. The American journal of pathology 162:575-586), Multimerin-1 (Roesli, C, et al. 2008. Faseb J),
  • Neuropilin-2 (Yuan, L., et al. 2002. Development 129 :4797-4806 / Caunt, M., et al. 2008.
  • Plakoglobin (Hirakawa S., et al. 2003. The American journal of pathology 162:575-586).
  • Proteinase-activated receptor-2 (Hirakawa S., et al. 2003. The American journal of pathology 162:575-586), Reelin (Hirakawa S., et al. 2003. The American journal of pathology 162:575-586),
  • VCAM-I Vascular cell adhesion molecule 1
  • VE-cadherin Vascular-endothelial cadherin (VE-cadherin) (Baluk, P., et al. ' 2007. The Journal of experimental medicine 204 -.2349-2362),
  • Vascular endothelial growth factor receptor-3 (VEGFR-3) (Kaipainen, A., et. al. 1995. Proceedings of the National Academy of Sciences of the United States of America
  • the at least one labeled antibody is a 124 I labeled antibody to the lymphatic epitope LYVE-I and the image is acquired using positron emission tomography.
  • the antibody is an IgG antibody.
  • the labeled antibody or labeled non-antibody binder is delivered to the patient in an amount to saturate the lymphatic vessels.
  • the present invention relates to a method for the making of labeled antibodies or labeled non-antibody binders to at least one non-intracellular, surface accessible protein present in or expressed on lymphatic vessels, or in the extracellular matrix of lymphatic vessels preferably for use in a as described above.
  • candidate proteins expressed by lymphatic endothelial cells are selected either from literature or by transcriptional or proteomic profiling of lymphatic endothelial cells, wherein preferably one of the candidate proteins systems as detailed above is selected
  • in a second step the staining patterns of antibodies or non-antibody binders to candidate proteins are evaluated on frozen tissue sections of test organs, including lymph nodes with lymphangiogenic lymphatic vessels, wherein, if high affinity monoclonal antibodies or high-affinity non-antibody binders against candidate proteins are not available, high affinity antibodies are produced preferably by antibody-phage display or hybridoma technology
  • antibodies or non-antibody binders that show preferential staining of lymphatic vessels or their extracellular matrix are selected and delivered to living organisms bearing lymph node lymphangiogenesis and then detected in tissue sections for evaluation whether they accumulate specifically in lymphatic vessels or their extracellular matrix compared to control antibodies or non-antibody binders, and whether they
  • a biodistribution analysis is performed to select for and quantify the in vivo targeting performance of the labeled antibodies or non-antibody binders to candidate proteins compared to labeled control antibodies or non-antibody binders in living test organisms bearing lymph node lymphangiogenesis and antibodies or non-antibody binders are selected, the activity concentration of which to candidate molecules in the lymph node with on-going lymph node lymphangiogenesis is higher, preferably at least twice as high, as in the adjacent tissues and as well preferably twice as high as the enrichment of the injected control antibody or non-antibody binder, optionally supplemented with the analysis of sections of organs adjacent to lymph nodes with ongoing lymphangiogenesis and control lymph nodes by microradiography to evaluate the enrichment pattern in these organs.
  • this method in particular but not limited to the situation of the case of positron emission tomography can be described for a more specific embodiment as follows:
  • candidate proteins need to be selected.
  • Candidate proteins are expressed by lymphatic endothelial cells (LECs), the cell type lining the lumen of lymphatic vessels. They are selected either from the literature or are identified by transcriptional or proteomic profiling of LECs as described below.
  • LECs lymphatic endothelial cells
  • LECs are isolated from mouse tissue by tissue digestion and fluorescence-activated cell sorting (FACS) as described (Halin, C, et al. 2007. Blood 110:3158-3167.). The transcriptional profile of LECs is evaluated by microarray analysis as described (Shin, J.W., et al. 2008. Blood 112:2318-2326.). Genes up-regulated on activated
  • LECs compared to resting LECs are identified by comparing the transcriptional profiles of LECs isolated from normal tissue and LECs from tissue containing activated LECs, e.g. inflamed or tumor-associated tissue.
  • resting or activated LECs can also be isolated from normal tissue or tissue with activated lymphatic vessels by laser capture microdissection (LCM) as described (Burgoon, M. Pet al. 2005. Proceedings of the National Academy of Sciences of the United States of America 102:7245-7250.) and analyzed by microarray analysis. Identification of candidate proteins by proteomic profiling: The surface accessible proteome of cultured LECs can be identified by two dimensional peptide mapping as described (Roesli, C, V.
  • Proteins up-regulated on activated compared to resting LECs are identified by comparing the surface accessible proteomes of LECs cultured with lymphangiogenic growth factors and LECs cultured in medium depleted of lymphangiogenic growth factors.
  • Candidate proteins should be surface accessible, i.e. expressed on the cell surface membrane, or be enriched in the extracellular matrix and must not be intracellular. They have to be consistently identified on LECs. Ideally, they are up-regulated on activated (i.e. expanding) compared to control LECs.
  • activated i.e. expanding
  • candidate proteins whose expression is detected predominantly on lymphatic vessels. It is probable that no protein is exclusively present on activated lymphatic vessels and has a non-redundant function. Thus candidates that fit these criteria as best are selected.
  • Candidate protein must not be strongly expressed on B or T cells or tissues adjacent to the lymph nodes that are aimed to be imaged in vivo.
  • the staining patterns of antibodies to candidate proteins are evaluated on frozen tissue sections of various mouse organs, including lymph nodes with lymphangiogenic lymphatic vessels. Sections of a mouse model of tumor- or inflammation-induced lymph node lymphangiogenesis can be used (Halin et al, 2007). If there are no suitable, high affinity monoclonal antibodies against candidate proteins available, they can be produced, for instance by antibody-phage display or hybridoma technology (Kohler, G., and C. Milstein. 1975. Nature 256:495-497; McCafferty, J., 1990, Nature 348:552-554).
  • the staining pattern of the antibodies should be at possible restricted to lymphatic vessels or their extracellular matrix. If an antibody stains other structures than lymphatic vessels, equally well, this may indicate either off-target binding of the antibody or expression of the target aside lymphatic vessels. To address the first issue, another antibody against the target protein with a higher specificity could be tested. The candidate protein is dismissed if distinction between the additionally stained structures and the lymphatic vessels in lymph nodes is unlikely to be achieved by in vivo imaging.
  • lymphatic vessels or their extracellular matrix are intravenously injected into mice bearing lymph node lymphangiogenesis.
  • lymph node lymphangiogenesis a mouse model of tumor- or inflammation-induced lymph node lymphangiogenesis can be used (Halin et al, 2007).
  • Injected antibodies to the candidate protein are detected in tissue sections and it is evaluated whether they accumulated specifically in lymphatic vessels or their extracellular matrix compared to control IgG, and whether they enrich in the activated lymphatic vessels or their extracellular matrix in the lymph nodes. If this is not the case, antibody dosing and time point of observation can be adjusted. If no enrichment in the lymphatic vessels or in their extracellular matrix is achieved, the candidate protein is dismissed.
  • an antibody that stains its target well in frozen tissue sections does not imperatively bind its target as well in vivo. If an antibody stains other structures than lymphatic vessels or their extracellular matrix equally well, this may indicate either off-target binding of the antibody or expression of the target aside lymphatic vessels.
  • antibodies are labeled, e.g. an I radionuclide such as radionuclide 125 I, and then tested whether they maintain their binding affinity. This can be done by evaluating the immunoreactivity on antigen loaded columns. Alternatively, if no antigen loaded columns are available, the antibodies are injected into mice. Then tissue sections of the mice are analyzed by microradiography to analyze whether the iodinated antibodies still accumulate in the lymphatic vessels or their extracellular matrix. In case an iodinated antibody does not bind to its target anymore, the iodination protocol is adjusted or another antibody is applied.
  • an I radionuclide such as radionuclide 125 I
  • Biodistribution analysis are performed to quantify the in vivo targeting performance of the 125 I-lableled antibodies to candidate proteins compared to l25 I-labeled control antibodies in mice bearing lymph node lymphangiogenesis.
  • the activity concentration of antibodies to candidate molecules in the lymph node with on-going lymph node lymphangiogenesis needs to be at least twice as high as in the adjacent tissues and as well twice as high as the enrichment of the injected control IgG.
  • accumulation in other organs than lymph nodes should be as low as the background of injected control antibody. Sections of organs adjacent to lymph nodes with on-going lymphangiogenesis and control lymph nodes are additionally analyzed by microradiography to evaluate the enrichment pattern in these organs.
  • Optimal doses and time points of imaging are characterized by target saturation in the lymph nodes with on-going lymphangiogenesis while other organs should have low activity concentrations.
  • antibodies are further tested for their ability to detect lymph node lymphangiogenesis in vivo by positron emission tomography (PET) imaging.
  • PET positron emission tomography
  • they are labeled with a radionuclide suitable for PET imaging (12).
  • radionuclide suitable for PET imaging (12).
  • the binding affinity of the labeled antibody has to be tested as described above.
  • Radiolabeled antibodies to candidate molecules and control antibody are injected into mice bearing lymph node lymphangiogenesis and PET imaging is performed. If specific signals at the sites of lymph nodes with expanded lymphatic networks can be visualized by PET imaging, the mice are sacrifized and re-scanned by PET with these lymph nodes placed next to them.
  • lymph nodes were imaged in vivo. If in in vivo PET scans the lymph node bearing expanded lymphatic networks gives a stronger signal than control lymph nodes, it is confirmed, that lymph node lymphangiogenesis is imaged.
  • lymph nodes cannot be imaged in vivo, the specific activity of the labeled antibodies may have been too low. In this case, the radiolabeling procedure can be adjusted. Furthermore, changing to a radionuclide with better resolution may help to increase contrast and resolution.
  • the present invention relates to the use of an antibody or labeled non-antibody binder as determined in a method as given above for non-invasive in vivo imaging of the lymphatic system, preferably by positron emission tomography. More specifically, the invention pertains to the use of an IgG based ' I labeled antibody to the lymphatic epitope LYVE-I for non-invasive in vivo imaging with positron emission tomography.
  • the invention in another embodiment relates to the use of a method as described above for imaging , preferably using a antibody or non-antibody binding system as made using a method as given above, for the detection of lymphangiogenesis, cancer and/or inflammation.
  • the invention furthermore pertains to the use of a method for imaging as given above, for the monitoring and the control (determination and/or adaptation) of the medical treatment such as cancer radioimmunotherapy of a patient.
  • the present invention relates to an IgG based 124 I labeled antibody to the lymphatic epitope LYVE-I in particular for non-invasive in vivo imaging with positron emission tomography.
  • lymphatic vessels can unexpectedly be targeted and imaged by antibodies and present for the first time the proof of non-invasive imaging of lymph node lymphangiogenesis in vivo.
  • This novel method may open up a future road of cancer metastasis diagnosis and cancer radioimmunotherapy, and may image lymphangiogenesis as a biomarker for the progression of the numerous medical conditions associated with lymphangiogenesis.
  • Harrell et al. (Harrell et al, American Journal of Pathology, 2007, 170: 774 - 786) imaged increased lymph flow correlating with lymph node lymphangiogenesis in tumor draining lymph nodes compared to control lymph nodes in a mouse tumor model. To this end, they injected tumor bearing mice interstitially with nanoparticles that were drained into the tumor draining lymph nodes and imaged flow of the nanoparticles non-invasively in vivo by near infrared imaging.
  • Ruddell et al. (Ruddell et al., Neoplasia, 2008, 10: 706-713) imaged increased lymph flow in tumor draining lymph nodes compared to control lymph nodes in the same mouse model as Harrell et al. They injected the mice interstitially with a contrast agent that was drained into the tumor draining lymph nodes. Following, the flow of the contrast agent was imaged non-invasively in vivo by magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • Harrell et al. and Ruddell et al. detected lymph node lymphangiogenesis only indirectly by measuring increased lymph flow.
  • tracer molecules were injected interstitially. In case lymphatic vessels were clogged by metastases it would not be possible to image lymph node lymhangiogenesis because the tracer molecules would not reach the lymph nodes.
  • molecules such as 124 I-labeled anti- LYVE-I antibody can be injected intravenously and reach the lymphatic vessels in the lymph nodes as well by extravasation of the blood vessels inside the lymph node and not only through tissue drainage.
  • lymph node lymphangiogenesis has been found to correlate with and even precede cancer metastasis.
  • lymph node lymphangiogenesis to be a novel biomarker for cancer metastasis. Imaging lymph node lymphangiogenesis non-invasively in vivo in cancer patients would circumvent sentinel or local lymph node dissection and its side effects and may even be more sensitive in detecting cancer metastasis than the currently applied method. Biomarker observation.
  • Many pathological conditions e.g.
  • lymphatic vasculature chronic inflammation, rheumatoid arthritis, and cancer
  • - Cancer treatment Many cancer types have been shown to induce lymphangiogenesis in and around the tumor. Radiolabeled antibodies to lymphatic endothelial cell specific epitopes like LYVE-I or molecules that are specifically up-regulated on growing lymphatic vessels may be used for radioimmunotherapy of cancers. Further embodiments of the present invention are outlined in the dependent claims.
  • Fig. 1 shows how a systemically injected radiolabeled anti-LYVE-1 antibody accumulates in the lymphatic vasculature;
  • a - N show microradiographies of tissue sections of 1 25 I-anti-LYVE-l antibody- and control IgG-injected mice; the radio signal of the injected l25 I-anti-LYVE-l antibody (A, C, E, G, I, K, M) but not of -control IgG (B, D, F, H, J, L, N) was detected in sections of control (A, B) and inflamed auricular lymph nodes (C, D), control (E, F) and inflamed ears (G, H), lung (I, J), and intestine (M, N); In liver sections (K, L), both 125 I-anti-LYVE-l antibody and control IgG could not be detected.
  • Fig. 2. shows the dose dependent accumulation of l25 I-anti-LYVE-l antibody in the lymph nodes; biodistribution experiments of different doses of l25 I-anti-LYVE-l antibody or -control IgG in mice; the results are expressed as the percentage of the injected radioactivity dose per gram of tissue (%ID/g) ⁇ SD; (A) seven microgram injected 1 25 I-anti-L YVE-I antibody resulted primarily in targeting of the lungs; (B) thirty- five microgram injected 125 I-anti-LYVE-l antibody resulted in increased targeting of lymph nodes compared to seven microgram injected dose; (C) ninety micrograms injected 125 I-anti-LYVE-l antibody did not further increase the targeting of the lymph nodes compared to other organs; enhanced accumulation of antibody in the blood compared to thirty-five microgram injected dose suggested that the lymph nodes were saturated with l25 I-anti-LYVE-l antibody.
  • Fig. 3. shows the accumulation of 125 I-anti-LYVE-l in the different organs dropped continuously over time; biodistribution experiments of 125 I-anti-LYVE-l antibody at day 1 (approximately 24 h), day 2 (approximately 48 h), and day 3 (approximately 72 h) after antibody injection; the results are expressed as the percentage of the injected radioactivity dose per gram of tissue (%ID/g) ⁇ SD; accumulation of injected I-anti-LYVE-1 antibody in the analyzed organs and the blood dropped uniformly over time; the data from day 1 after antibody injection were from Figure 2;
  • Fig. 4. shows in vivo imaging of inflammation-induced lymph node lymphangio genesis by PET using l24 I-anti-LYVE-l antibody;
  • A, B maximal intensity projections (MIP) of in vivo scanned mice injected with l24 I-anti-LYVE-l antibody (A) or -control IgG (B);
  • A The inflamed auricular lymph node with on-going lymphangiogenesis (black arrow) accumulated more 124 I-anti-LYVE-l antibody than the contra-lateral control auricular lymph node (grey arrow); brachial and axillary lymph nodes were as well clearly visible (arrow heads);
  • B in vivo PET image of an 124 I-control IgG injected mouse; most of the antibody accumulated in the blood pool and visualized the heart (black arrow);
  • C, D in order to compare antibody accumulation in lymph nodes of ' I-anti-LYVE-1 antibody (C) and -control IgG (D) injected mice,
  • Fig. 5. shows the accumulation of 124 I-anti-LYVE-l antibody in the lymphatic vessels of the lymph node; sections of an inflamed auricular lymph node of a 124 I-anti-LYVE-
  • Fig. 6. shows in vivo PET imaging of tumor-induced lymphangiogenesis in popliteal lymph nodes using 124 I-anti-LYVE-l antibody,
  • (a) The tumor draining popliteal lymph node with on-going lymphangiogenesis (black arrow) is clearly visible, in contrast to the contra-lateral control popliteal lymph node
  • lymph node lymphangiogenesis establishes a method to image lymph node lymphangiogenesis non-invasively.
  • systemically injected antibodies to lymphatic epitopes accumulate in the lymphatic vasculature in tissues and lymph nodes, determined by biodistribution and microradiography analyses.
  • lymphatic vessel endothelial hyaluronan receptor- 1 LYVE-I
  • a systemically injected antibody to a lymphatic epitope accumulates in the lymphatic vasculature: To assess whether antibodies can be used to target and image lymphatic vessels in vivo, it was first tested whether following systemic injection of an antibody against a lymphatic-specific epitope, the antibody accumulates in the lymphatic vasculature. Since antibodies are large-sized molecules (150 kD), leaky blood vessels, which are a hallmark of tumors and inflammation, can promote the antibodies' extravasation from blood vessels and consecutive uptake by and accumulation in the lymphatic vessels. Therefore, an established mouse model of chronic skin inflammation was used.
  • K14/VEGF transgenic mice are subjected to a cutaneous delayed- type hypersensitivity reaction, induced by topical application of the contact sensitizer oxazolone to the skin.
  • Kl 4/VEGF mice develop a chronic skin inflammation that is associated with vascular hyperpermeability, prominent lymphangiogenesis in the skin and, in particular, in the draining lymph nodes.
  • the Kl 4/VEGF mice were first given systemic injections of a rat antibody to the vascular endothelial growth factor receptor-3 (VEGFR-3) that is expressed by lymphatic endothelium or a control immunoglobulin (Ig)G.
  • VAGFR-3 vascular endothelial growth factor receptor-3
  • mice were killed and tissue sections were stained with a fluorescently labeled secondary antibody against rat IgG.
  • the anti-VEGFR-3 antibody was specifically detected on lymphatic vessels in the skin, lung, intestine, and tongue; there were high levels of reactivity with the inflamed and control draining auricular lymph nodes.
  • the VEGFR-3 immunoreactivity co-localized with that of the lymphatic marker LYVE-I .
  • VEGFR-3 was not detected on blood vessels that were strongly positive for the blood vessel marker Meca32.
  • the injected control IgG was not detected on lymphatic vessels and only showed a weak and diffuse staining of LYVE-I- negative areas in the lymph nodes. So, the injected anti-VEGFR-3 antibody specifically accumulated in the lymphatic vessels; surprisingly, normal blood vessels in organs such as the skin and the intestine did not prevent antibody extravasation, thus enabling the detection of lymphatic vessel even in healthy organs.
  • Iodination of anti-VEGFR-3 antibody does not inhibit its binding to lymphatic vessels in vivo: It was aimed to use antibodies against lymphatic epitopes that were labeled with the radionuclide 125 I in the biodistribution experiments. However, iodination of antibodies can decrease their binding affinity for antigen. Thus, it was next investigated whether a systemically injected l25 I-anti- VEGFR-3 antibody accumulates in the lymphatic vessels of the Kl 4/VEGF mice with unilateral lymphangiogenesis. Microradiography of tissue sections obtained 48 h after antibody injection into mice revealed strong localization of the 125 I-anti- VEGFR-3 antibody at vessel-like structures in the skin and in the draining lymph nodes.
  • Lymphatic-specific expression of the injected anti- LYVE-1 antibody was also detected in other organs, including the intestine, tongue, and salivary glands.
  • the anti-LYVE-1 antibody did not co-localize with blood vessels that strongly expressed the vascular marker Meca32.
  • the injected rat IgG control antibody was not detected in lymph nodes, skin or other organs. These results indicate that the anti-LYVE-1 antibody accumulates specifically in the lymphatic vasculature of inflamed and normal tissue.
  • the rat anti-LYVE-1 antibody and control rat IgG were radiolabeled with 125 I and injected systemically into K14/VEGF mice bearing unilateral lymph node lymphangiogenesis.
  • the injected radiolabeled anti-LYVE-1 antibody but not the injected radiolabeled rat IgG, was clearly detected by microradiography of tissue sections from auricular lymph nodes, ears, lungs, and intestines • (Fig. 1) and was localized at vessel-like structures.
  • LYVE-I staining obtained with a rabbit antibody overlapped with the radio signal of the injected anti-LYVE-1 antibody on serial sections of the ears and auricular lymph nodes (Fig. l),indicating that the affinity of anti- LYVE-1 antibody for its target was not inhibited by radioiodination.
  • Radiolabeled anti-LYVE-1 antibody accumulates in a dose- dependent manner in the lymphatic vessels of the lymph nodes: Biodistribution experiments were performed with the l25 I-anti-LYVE-l antibody and l25 I-control rat IgG to evaluate their in vivo targeting to sites of lymphangiogenesis within lymph nodes. To this end, different amounts of radiolabeled antibodies (7 ⁇ g, 35 ⁇ g, or 90 ⁇ g) were injected intravenously into K14/VEGF mice with unilateral, inflammation-induced lymph node lymphangiogenesis. At 1 day after injection of 7 ⁇ g anti-LYVE-1 antibody, preferential accumulation of the antibody in the lung (89% of the injected dose [ID]/g of tissue) was found (Fig.
  • mice injected with control IgG concentrations of radioactivity in the lymph nodes (between 2%-3 %ID/g) were less than in mice injected with the anti-LYVE-1 antibody, indicating the specificity of the anti-LYVE-1 antibody for lymphatic tissues.
  • concentrations of radioactivity in the lymph nodes between 2%-3 %ID/g were less than in mice injected with the anti-LYVE-1 antibody, indicating the specificity of the anti-LYVE-1 antibody for lymphatic tissues.
  • Increasing the amount of injected anti-LYVE-1 antibody from 35 ⁇ g to 90 ⁇ g did not increase the uptake of anti- LYVE-1 antibody in the lymph nodes, compared to other organs, but increased the amount of radioactivity measured in the blood, indicating saturation of the target (Fig. 2 C and Table Sl).
  • Table Sl Dose dependent accumulation of 125 I-anti-LYVE-l antibody in the lymph nodes. Biodistribution experiments of different doses of 125 I-anti-LYVE-l antibody or -control IgG in mice. The results are expressed as the percentage of the injected radioactivity dose per gram of tissue (%ID/g) ⁇ SD.
  • Table S2 Increase in weight and radioactivity in auricular lymph nodes upon inflammation Upon inflammation weight and radioactivity of the auricular lymph nodes were increased. Data are derived of the biodistribution analysis of Figure 2.
  • microradiograpy confirmes that the l25 I-anti-LYVE-l antibody is specifically enriched in lymphatic vessels at all three timepoints.
  • the auricular lymph node/blood ratios increased approximately 3-fold by 2 days after injection and 5-fold thereby 3 days, compared to with the first day after injection (Fig. 3, Table S3 and Table 1);
  • day 1 is chosen as the best timepoint for the consecutive in vivo imaging studies.
  • Table 1 Accumulation of radiolabeled anti-LYVE-1 antibody in the auricular lymph nodes compared to adjacent tissues and the blood.
  • results are expressed as the percentage of the injected radioactive dose per gram of tissue (%ID/g) ⁇ SD
  • Table S3 Accumulation of I25 I-anti-LYVE-l in the different organs drops continuously over time. Biodistribution experiments of ' s I-anti-LYVE-l antibody at day one (approximately 24 h), day two (approximately 48 h), and at day three (approximately 72 h) 5 after antibody injection.
  • lymph node lymphangiogenesis in vivo by PET imaging Based on the encouraging biodistribution results, it was verified that it is possible to visualize lymphatic vessels within lymph nodes by in vivo PET imaging of mice.
  • the anti-LYVE-10 antibody and control rat IgG were radiolabeled with the positron emitter 124 I.
  • 124 I and 125 I are isotopes that have identical chemical behaviors, so the results obtained from the biodistribution analysis with 125 I-anti-LYVE-l antibody could be used to determine the amount of l24 I-anti-LYVE-l antibody for injection and the timepoint for in vivo imaging.
  • mice were injected 38 ⁇ g of the radiolabeled anti-LYVE-1 antibody or control IgG intravenously5 into K14/VEGF mice bearing unilateral lymph node lymphangiogenesis.
  • the mice were scanned by PET to obtain in vivo images.
  • I-anti-LYVE-l antibody strong radio signals were produced at the sites of the auricular, brachial and axillary lymph nodes (Fig. 4 A).
  • the accumulation of 24 I-anti-LYVE-l antibodies in other organs was similar or weaker, compared to the lymph nodes sites. In particular, there was a low level of accumulation in liver, kidney and intestine.
  • the imaged animals were sacrificed and imaged, ex vivo, by PET, with the dissected auricular lymph nodes placed next to the heads of the mice. Coronal sections of these data sets were normalized to compare them with the in vivo sections.
  • the strong radio signals that were detected in vivo in the regions of the auricular lymph nodes were no longer detectable in the sections of the ex vivo scans (Fig. 4 E). Instead, the auricular lymph nodes that were placed next to the head of the animals emitted a strong radio signal (Fig. 4 F).
  • the radio signals from the regions of the axillary and brachial lymph nodes in the corresponding sections were comparable between the in vivo and ex vivo PET scans (Fig. 4 C and F).
  • ex vivo scans of mice injected with the control IgG there were no detectable signals in the dissected lymph nodes.
  • lymph node lymphangiogenesis within auricular lymph nodes can be imaged in vivo by PET following systemic injection of a radiolabeled, lymphatic-specific antibody.
  • lymphatic vessels can be targeted and imaged by antibodies
  • proof-of-principle is provided for the non-invasive in vivo imaging of lymph node lymphangiogenesis by PET.
  • VEGFR-3 has been detected on subsets of activated macrophages, monocytes and dendritic cells, and is expressed by blood vascular endothelial cells during embryogenesis, and, likely, on some angiogenic blood vessels associated with tumors and wounds, the combined microautoradiography and immunofluorescence studies reveal that an injected anti- VEGFR-3 antibody specifically accumulates in LYVE-I -positive lymphatic vessels of the skin and the lymph nodes, but not in Meca32-positive blood vessels.
  • the accumulation of injected anti- VEGFR-3 antibody is not caused by unspecific uptake of Igs by lymphatic vessels, because injection of equal amount of control IgG does not lead to any detectable accumulation in lymphatic vessels.
  • circulating anti- VEGFR-3 antibodies seem to be extravasated from leaky blood vessels at sites of inflammation and drained by lymphatic vessels, where they bound to VEGFR-3 expressed on the luminal surface.
  • an intraperitoneally injected anti-ICAM-1 antibody was recently detected by immunofluorescence analysis of inflamed lymphatic vessels.
  • an antibody against LYVE-I a hyaluronan receptor that is specifically expressed by lymphatic endothelium but not by blood vessels, is used.
  • LYVE-I Apart from its lymphatic expression, LYVE-I is only present on liver sinusoidal endothelial cells and a subset of macrophages and embryonic blood vessels. Most importantly, however, no non- redundant function of LYVE-I has been identified, despite intense research on this most widely used marker for lymphatic vessels.
  • the anti-LYVE-1 antibody efficiently accumulates in lymphatic vessels of different organs and tissues (skin, lymph nodes, lung, intestine), indicating bioavailability comparable with that of the anti- VEGFR- 3 antibody.
  • lymph node lymphangiogenesis has been identified as an early marker of metastasis to lymph nodes in experimental models.
  • expansion of lymphatic networks in sentinel lymph nodes has also been observed in patients with melanoma or breast cancer and found to be a significant predictor of distant metastasis.
  • the use of PET imaging of radiolabeled antibodies, in particular of anti-LYVEl antibodies, to detect lymph node lymphangiogenesis represents a less invasive, simpler and potentially more sensitive method to identify patients with lymph node metastases than current approaches, including sentinel lymph node dissection.
  • the method also avoids of the need to inject dyes around tumors; this technique does not always lead to the detection of all draining lymph nodes, due to the location of the injection or the clogging of lymphatic vessels by metastatic tumor cells.
  • immuno-PET using l24 I-anti-LYVE-l offers flexibility in time of image collection; it is e.g. possible to image lymph node lymphangiogenesis 2 days after antibody injection.
  • Immuno-PET imaging of lymph node lymphangiogenesis can be applied to medical fields beyond oncology, because many pathological conditions (e.g. chronic inflammatory diseases including rheumatoid arthritis) are associated with lymphangiogenesis.
  • pathological conditions e.g. chronic inflammatory diseases including rheumatoid arthritis
  • lymphangiogenesis can be imaged and used as a biomarker for disease progression or response to therapy.
  • Radiolabeled antibodies against lymphatic endothelial cell epitopes such as LYVE-I or other molecules that are specifically up-regulated on growing lymphatic vessels can also be used for radioimmunotherapy of metastases associated with tumor lymphangiogenesis.
  • mice were sensitized by topical application of a 2% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; Sigma- Aldrich, St. Louis, MO, USA) solution in acetone/olive oil (4:1 vol/vol) to the shaved abdomen (50 ⁇ l) and to each paw (5 ⁇ l) as described.
  • acetone/olive oil 4:1 vol/vol
  • the right ears were challenged by topical application of 20 ⁇ l of a 1% oxazolone solution.
  • the resulting inflammation in the ear and the ear draining (auricular) lymph node is accompanied by lymphangiogenesis. All animal experiments were approved by the cantonal veterinarian office Zurich.
  • B16-F1 murine melanoma cells (kindly provided by Dr. S. Hemmi, University of Zurich, Switzerland) were cultured in D-MEM (Invitrogen, Carlsbad, CA, USA) containing 10% FBS and were transfected by electroporation with fiill-length human-VEGF-C subcloned into the pcDNA3.1 (Invitrogen) vector. Stable clones (B 16-Fl -VEGF-C cells) were selected and VEGF-C expression was confirmed by RT-PCR and ELISA.
  • Ex vivo fluorescence experiments Four hundred and fifty micrograms of rat anti -mouse VEGFR-3 antibody (mF4-31Cl) or rat control IgG were injected intraperitoneally into 10- week-old C57BL/6J K14/VEGF transgenic mice (one mouse per treatment), one day after challenging one ear with oxazolone. Forty-eight hours after injection, the animals were sacrificed and organs were frozen in optimal cutting temperature (OCT) compound (Sakura Finetec, Zoeterwoude, Netherlands).
  • OCT optimal cutting temperature
  • rat anti- mouse LYVE-I antibody (clone 223322, R&D Systems, Minneapolis, MN, USA) or isotype-matched rat control IgG were injected into the tail veins of 10- week-old K14/VEGF mice (one mouse per treatment) 1 day after challenging one ear with oxazolone. Twenty- four hours after injection, the animals were sacrificed and organs were frozen in OCT compound. Seven-micrometer frozen sections were cut and fixed with 4% paraformaldehyde in PBS for 15 min at 4°C.
  • the sections were incubated with an Alexa Fluor 594 conjugated donkey anti-rat IgG antibody (Invitrogen, Carlsbad, CA, USA).
  • the sections were co-stained with a rabbit anti- mouse LYVE-I antibody (Angiobio, Del Mar, CA, USA) that was detected by an Alexa Fluor 488 donkey anti-rabbit IgG antibody (Invitrogen), or co-stained with a biotinylated rat Meca32 antibody (specific staining of blood vessels; BD Pharmingen, Franklin Lakes, New Jersey, USA) that was detected by Alexa Fluor 488 streptavidin (Invitrogen).
  • Antibodies were radiolabeled with Na 125 I (Perkin Elmer, Waltham, MA, USA) by adapting the standard chloramine-T method. Briefly, 70 - 380 ⁇ Ci Na I and 5 ⁇ l of 5 mg/ml chloramine T (Sigma- Aldrich) were added per 100 ⁇ g antibody in PBS, concentrated to 1 mg/ml. After 2 minutes, the radiolabeled antibodies were separated from free 125 I using PDlO columns (GE-Healthcare, Chalfont St. Giles, UK) pretreated with 1 ml 0.1% BSA and equilibrated in PBS. The radioactivity of the samples was determined using a ⁇ -counter (Cobra Autogamma, Packard Instrument Comp., Meriden, CT, USA).
  • Radiolabeled iodine Normal uptake of radiolabeled iodine by the thyroid glands was blocked by administration of potassium iodide (Fluka, Buchs, Switzerland) in the drinking water starting four days before an experiment. Binding of radiolabeled iodine by iodine symporters in the intestine was blocked by oral administration of sodium perchlorate (Fluka) one hour before antibody injection.
  • mice Forty-eight hours after injection, mice were sacrificed and organs, including lymph nodes, were frozen in OCT compound.
  • Anti- LYVE-1 antibody 35 ⁇ g, 150 ⁇ Ci
  • rat control IgG 35 ⁇ g, 120 ⁇ Ci
  • Seven-micrometer frozen sections of the tissues were fixed with 4% paraformaldehyde in PBS at 4°C for 15 min.
  • Air-dried sections were coated with KODAK autoradiography emulsion type NTB (CARESTREAM HEALTH, INC., Rochester, NY, USA), dried and stored at room temperature for 2 weeks ( 125 I-Or 124 I- anti-LYVE-1 and corresponding control IgG sections) or for 3 weeks (' 5 I-anti-VEGFR-3 and corresponding control IgG sections) as described (45).
  • the sections were developed in KODAK Developer Dl 9 (C ARESTREAM HEALTH, INC.) for 4 min and fixed with RA 3000 fixer (Kodak) for 5 min. Finally, the sections taken from mice that had received injections of anti-VEGFR-3 or the corresponding control IgG were counterstained with hematoxylin (Richard Allan Scientific, Kalamazoo, MI, USA).
  • l24 I-anti- LYVE-I 38 ⁇ g, 0.37-0.42 mCi
  • 124 I-labelled rat control IgG 38 ⁇ g, 0.34-0.36 mCi
  • Injected doses were quantified by measuring syringes before and after injection using a VEENSTRA dose calibrator (Siemens, Er Weg, Germany). Normal uptake of radiolabeled iodine by the thyroid glands was blocked by administration of potassium iodide starting four days prior to antibody injection and the mice were given sodium perchlorate perorally one hour before antibody injection.
  • PET scans were performed approximately 24 h (2 mice per group) or 48 h (1 mouse per group) after intravenous radiotracer injection using the GE Vista/CT camera (GE Healthcare) as described previously.
  • mice were anesthesized with isoflurane (Abbott Laboratories, Abbott Park, IL, USA) in an air/oxygen mixture and monitored during PET scanning as described previously. After termination of the acquisition, mice were sacrificed by cervical dislocation. Auricular lymph nodes were dissected and the mice were re-scanned, with the dissected auricular lymph nodes positioned next to their heads. Positrons emitted by ' I move in average 2.3 mm away from their source until they annihilate with an electron.
  • lymph nodes are only a few cubic millimeters in size, accumulation of radiotracer in isolated extracted lymph nodes can be missed, because there is little mass for positron slowdown.
  • the dissected lymph nodes were placed in agar blocks to surround them with mass.
  • whole-body PET data were acquired in two bed positions (30 min acquisition time per bed position) and were reconstructed in a single timeframe, with pixel sizes of 0.3875 mm and 0.775 mm in the transverse and axial directions, respectively.
  • Series of coronal image slices and maximum intensity projections (MIPs) as well as MIP movies, were generated using the dedicated software PMOD (PMOD Technologies Ltd., Adliswil, Switzerland). MIPs render three-dimensional images into two dimensions by displaying the most intensive value from each voxel stack. For visual inspection and comparison of antibody uptake in different mice, PET image sections were displayed with a fixed grey scale normalized to the injected dose per body weight.
  • 124 I- anti-LYVE-1 (30 ⁇ g, 0.26-0.38 mCi) or 124 I-labelled rat control IgG (30 ⁇ g, 0.32-0.33 mCi) were injected intravenously into tumor bearing mice 19 . days after tumor cell injection. PET imaging and biodistribution analysis of selected organs were performed as described above.

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Abstract

La présente invention concerne un procédé d'imagerie non invasive in vivo du système lymphatique, en particulier de la lymphangiogenèse ainsi que des procédés de fabrication d'un anticorps marqué destiné à être utilisé dans de tels procédés ainsi que l'utilisation d'un tel anticorps marqué. Le procédé proposé comprend les étapes consistant à administrer au moins un anticorps marqué à l'au moins une protéine non intracellulaire, accessible en surface, présente dans, ou exprimée sur les vaisseaux lymphatiques, ou présente dans la matrice extracellulaire des vaisseaux lymphatiques, à un patient; permettre à l'au moins un anticorps marqué d'être distribué et éventuellement de s'accumuler dans le système lymphatique; acquérir au moins une image associée au marqueur de l'anticorps marqué du système lymphatique.
PCT/EP2010/001044 2009-02-25 2010-02-19 Procédé d'imagerie in vivo de la lymphangiogenèse de nœuds lymphoïdes par tomographie par émission d'immuno-positrons et marqueurs pour celle-ci Ceased WO2010097182A1 (fr)

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CN115531558A (zh) * 2022-09-16 2022-12-30 华中科技大学 一种动物肝脏淋巴管系统的标记和三维图谱成像方法
CN116236577A (zh) * 2023-02-20 2023-06-09 河南师范大学 神经导向因子Sema3A的应用及治疗骨溶解症的药物组合物

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109952312A (zh) * 2016-09-29 2019-06-28 纳塞恩特公司 生腱蛋白表位和其抗体
WO2021068962A1 (fr) * 2019-10-11 2021-04-15 田中纯美 Polypeptide pour maladies liées à l'angiogenèse et à la lymphangiogenèse et utilisation correspondante
CN115404213A (zh) * 2022-09-16 2022-11-29 华中科技大学 一种肝脏淋巴管内皮细胞的分选方法、应用及试剂盒
CN115531558A (zh) * 2022-09-16 2022-12-30 华中科技大学 一种动物肝脏淋巴管系统的标记和三维图谱成像方法
CN116236577A (zh) * 2023-02-20 2023-06-09 河南师范大学 神经导向因子Sema3A的应用及治疗骨溶解症的药物组合物

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