title: Identifizierung und Charakterisierung FAP-spezifischer Liganden für die zielgerichtete Diagnostik und Therapie maligner Tumoren
creator: Loktev, Anastasia
subject: 500
subject: 500 Natural sciences and mathematics
subject: 570
subject: 570 Life sciences
subject: 610
subject: 610 Medical sciences Medicine
description: The identification of novel tumor-specific ligands as transport vehicles for both diagnostic and therapeutic radionuclides represents a major objective in nuclear medical oncology research. The use of selective compounds with high affinity to their target structure results in an increased tumor uptake of the radionuclide, while minimizing undesired radiation exposure of healthy tissue. Based on increasing knowledge about tumor physiology as well as ongoing technological progress, numerous tumor-targeting compounds have been identified and transferred into clinical practice. However, the majority of these compounds comprises antibodies, which feature serious deficiencies, such as poor membrane permeability and tissue penetration, slow clearance and immunogenicity. Peptides, instead, represent a favorable alternative with regard to size, pharmacokinetics and the possibility to be produced  cost-effectively in large quantities by automated solid phase synthesis.    Phage display technology represents a powerful tool for the de novo identification of target-specific peptides. This technique is based on the presentation of large polypeptide libraries on the surface of bacteriophages. To this end, the phage genome is manipulated to display the foreign peptides, which are physically linked to their encoding nucleic acids. Following several iterative rounds of incubation with any target protein of interest, the enriched target-specific ligands can therefore be easily identified by DNA sequencing.    Tumor growth and malignancy are not only affected by the characteristics of cancer cells but strongly depend on the attributes of several different cell types within the local tumor microenvironment. Cancer-associated fibroblasts (CAFs) represent an important subpopulation of these stromal cells and are known to promote tumor growth, inflammation and metastasis. They account for a large part of the tumor mass and are genetically more stable and therefore less susceptible to the development of therapy resistance than cancer cells per se. In contrast to normal fibroblasts, CAFs entail a number of tumor-specific marker proteins such as the integral membrane peptidase Fibroblast Activation Protein (FAP). This protein is highly expressed in the microenvironment of more than 90% of epithelial tumors including breast, lung and pancreatic carcinoma. Overexpression of FAP is associated with a poor prognosis in a wide range of different cancers. Due to very low expression levels in normal tissues, FAP represents an attractive molecular target for the development of tumor-specific diagnostics and therapies.    To identify novel FAP-specific peptides using phage display, two combinatorial libraries based on the scaffold structures Sunflower Trypsin Inhibitor-1 (SFTI-1) and Min-23 were built. They comprise a cyclic, disulfide-bridged backbone and a variable sequence consisting of six to ten random amino acids. After incubation of the libraries with the target protein in four rounds of biopanning, the FAP-specific ligands were isolated and their sequence determined using next-generation sequencing. A selection of suitable peptides was synthesized by solid phase synthesis, purified and characterized in different cell-based radioligand assays.    In contrast to the SFTI-1 based biopanning, which did not lead to any enrichment of FAP-specific peptides, the use of the Min-23 library was successful in identifying several FAP-targeting ligands. However, these peptides showed a very low affinity to their target protein, which most likely results from a rapid degradation. It is conceivable that the enzymatic function of the membrane protein effectuates a rapid hydrolysation of the small peptides upon initial binding. A solution to this is the use of an alternative scaffold, which impedes enzymatic cleavage or ensures cell internalization after hydrolysation.    In this context, the cyclotide-based FAP-specific miniprotein MC-FA-012, identified by BionTech AG, was characterized regarding target affinity, specificity and pharmacokinetic profile in radioligand binding assays as well as in FAP-positive tumor-bearing mice. In contrast to the Min-23-based peptides, MC-FA-012 and its trimerized version DOTA-(MC-FA-012)3 demonstrated high affinity to human FAP, while no substantial binding to the structurally related protein CD26 was observed. In addition, DOTA-(MC-FA-012)3 rapidly accumulated in FAP-positive tumor xenografts in vivo and showed negligibly low unspecific binding in healthy tissue, except for the kidneys. A first clinical analysis of the radiolabeled compound in patients with metastatic pancreatic carcinoma revealed a robust accumulation of the tracer in the primary tumor as well as in lymph node and bone metastases. In contrast, tracer uptake into normal tissue was very low. The radioactivity was cleared rapidly from the blood stream and excreted predominantly via the kidneys, resulting in high contrast PET images.    Since the high accumulation of DOTA-(MC-FA-012)3 in the kidneys could not be sufficiently reduced by different competition strategies, further development of the radiotracer regarding a potential therapeutic application was not pursued. The investigation of the structure-activity relations of MC-FA-012 revealed that both the scaffold and certain functional groups within the FAP-specific binding sequence account for high FAP affinity. Introducing even minor changes in the molecule's conformation resulted in an almost complete loss of binding.    Consequently, a different approach towards the development of a FAP-specific compound was adopted by designing the small molecules FAPI-01 to FAPI-15 based on a potent FAP inhibitor. Each of the compounds demonstrated high specific binding to human and murine FAP and internalized rapidly into FAP-expressing cells. Imaging and biodistribution studies in xenotransplanted mice proved a rapid tumor uptake of the tracers in a genetically modified FAP-overexpressing tumor model as well as in human FAP-negative tumors due to endogenous murine FAP expression. Notably, the radiotracers demonstrated rapid renal clearance without substantial binding to non-cancerous tissue. Due to their advantageous pharmacokinetics and an excellent stability in human serum, the compounds FAPI-02 and FAPI-04 were selected for further clinical investigation. Using PET/CT, biodistribution of the radiotracers was analyzed in patients with metastasized epithelial cancers, including breast, lung, pancreatic and colon carcinoma as well as in high-grade glioblastomas. Both tracers rapidly accumulated in the primary tumor as well as in soft tissue, lymph node and bone metastases. In contrast, tracer uptake into normal tissue was very low, resulting in favorable tumor-to-organ ratios and high contrast images.    Comparative imaging in one patient with locally advanced lung adenocarcinoma revealed an obvious advantage of FAPI-02 compared to the commonly used PET tracer 18F-FDG. FAPI-02 showed a higher tumor uptake and significantly lower activities in blood and liver, resulting in higher image contrast with better visibility of metastatic lesions. In contrast to FDG, which highly accumulates in cells with high glucose consumption, e.g. the brain or inflammatory tissue, FAPI-02 selectively targets FAP-expressing carcinomatous lesions. This opens up new perspectives for the precise diagnosis of malignant lesions in tissues with high metabolic activity, such as liver or brain.    Furthermore, various approaches for a potential therapeutic application of the FAP-ligands are currently being investigated, including different radionuclides and alternative effector molecules, such as chemotherapeutics or immunomodulators. Given the possibility to use either diagnostic or therapeutic nuclides with the same molecule, FAPI-02 allows simple stratification of patient cohorts likely to benefit from a therapeutic intervention.
date: 2018
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/25155/1/Loktev_DA_final_pdfa.pdf
identifier: DOI:10.11588/heidok.00025155
identifier: urn:nbn:de:bsz:16-heidok-251556
identifier:   Loktev, Anastasia  (2018) Identifizierung und Charakterisierung FAP-spezifischer Liganden für die zielgerichtete Diagnostik und Therapie maligner Tumoren.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/25155/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: ger