Advanced Therapeutic Technologies PDF

ADVANCED THERAPEUTIC TECHNOLOGIES Lesson 7 26/11/24 Theranostic strategies: new frontiers in precision medicine Theranostic is a term with multiple definitions and interpretations, first used in 1998. Etym...

ADVANCED THERAPEUTIC TECHNOLOGIES Lesson 7 26/11/24 Theranostic strategies: new frontiers in precision medicine Theranostic is a term with multiple definitions and interpretations, first used in 1998. Etymologically “theranostic” simply summarizes what has always been the goal of medicine, the inseparability of diagnosis and therapy, the pillars of medicine. Theranostics with radioligands (radiotheranostics) has played a pivotal role in oncology. Radiotheranostics explores the molecular targets expressed on tumor cells to target them for imaging and therapy. In this way, radiotheranostics entails non-invasive demonstration of the in vivo expression of a molecular target of interest through imaging, followed by the administration of therapeutic radioligand targeting the tumor- expressed molecular target. Therefore, radiotheranostics ensures that only patients with a high likelihood of response are treated with a particular radiotheranostic agent, ensuring the delivery of personalized care to cancer patients. (I took this part from an article: https://pubmed.ncbi.nlm.nih.gov/38555542/) RADIOLIGAND THERANOSTIC (RT) → Disease related biomarker (eg. Enzyme, receptor, transporter) + delivery of radioactive compound, that can be seen through molecular imaging (MI) techniques. Theranostic in nuclear medicine is currently one of the leading medical fields promoting the development of theranostics. In nuclear medicine, it refers to the concept of combined targeted imaging and therapy delivery. The binding part of a radiopharmaceutical is targeted to a specific structure on tumor cells. The targeting vector is labeled with radionuclide for Positron Emission Tomography (PET) or a single photon emission computed tomography (SPECT) for the purpose of characterization of the tumor tissue and for the confirmation of presence of the target molecule in the tumor mass. Therefore, this approach allows us to “see what we treat” and “treat what we see” at the molecular level. Historical timeline of the main milestones in radionuclide therapies and theranostics: - 1913 → Frederick Proescher publishes the first study on the therapeutic uses of intravenous radium - 1936 → John H. Lawrence uses phosphorous-32 to treat patients with leukemia (first clinical therapeutic application) - 1941 → Saul Hertz administers the first therapeutic dose of iodine-131. He is considered the pioneer of the field, establishing a historical landmark with the first clinical use of iodine-131 in patients with hyperthyroidism. - 1946 → Seidlin, Marinelli and Oshry treat a thyroid cancer with an atomic cocktail of iodine-131 - 1951 → FDA approves iodine-131NaI in thyroid patients; it is the first radiopharmaceutical ever approved - B Ansell and B.M Cook employ radiocolloids for the first studies on radiation synovectomy. After decades from the development of iodine-131, a breath of innovation came from the promising results of [177Lu]Lu-DOTATATE for the treatment of somatostatin receptor-positive gastro-entero-pancreatic neuroendocrine tumors and [177Lu]Lu-Prostate-Specific Membrane Antigen (PSMA) 617, for metastatic castration resistant prostate cancer. (Foto timeline slide 8 pdf. Theranostics, non l’ho messa perchè non so quanto sia importante ricordare tutte queste date) 1 Recent studies and advances in nuclear medicine have demonstrated the leading role of radiotheranostics in oncology, particularly in the diagnosis and treatment of malignant tumors using radioactive isotopes. They are used in oncology for several strategies: 1. Direct visualization and quantification of target expression using a single radiolabelled compound for diagnosis and therapy without altering the expression of the target; 2. Theranostic pairs, combining two radiopharmaceuticals that share the same structure and target but are differentially labelled with matching radioisotope pairs, allowing separate diagnosis and therapy; 3. Indirect imaging using reporter gene technology; 4. Imaging of downstream effects of gene and cell-based therapies. Direct visualization of target expression and therapy response Tracer accumulation and localization are directly related to the interaction of the tracer with its target, which in most cases is a receptor, transporter, enzyme or cell surface protein. The prototypical example is radioiodine treatment of thyroid diseases, Iodine- 131, a β-particle-emitting radiohalogen with a half- life of 8.02 days. Theranostic pairs Diagnostic and therapeutic radiopharmaceuticals that access the same cellular or biological process with paired radiolabels. This strategy has emerged for different cancer types, including neuroendocrine tumor (NET), prostate cancer, glioblastoma and bone metastases. Theranostic gene and cell therapy is a strategy developed to reduce the use of expensive, ineffective treatments or to replace them with more effective therapies at earlier time points. An example of this approach is CAR-T therapy, which is not only a gene and cell therapy but also an immunotherapy. This innovative approach reprograms the immune system to attack cancer. Specifically, T-cells are able to recognize and kill abnormal cells, such as those infected with viruses or cancer cells. Unfortunately, cancer cells find ways to evade the immune system, so the immune system needs to be retrained to recognize and attack cancer cells. This new strategy is based on the use of CARs (chimeric antigen receptors), which harness the normal function of T cells. The strategy, which is mainly used to treat liquid tumors, involves isolating T cells from the patient (leukapheresis), which are then engineered in the laboratory using vectors (mainly lentiviruses) that allow the expression of CAR on the surface of the cell. Once obtained, CAR-T cells are infused into the patient, where they recognize and bind the specific antigen expressed on the cancer cells surface. This «immunological synapse» leads to T- cell activation which in turn is responsible for cytolytic response and cancer cells death. Autologous T cells genetically engineered to express chimeric antigen receptor (CAR) have shown promising outcomes and emerged as a new curative option for hematological malignancy, especially malignant neoplasm of B cells such as B-ALL and lymphoma. Conversely, several clinical trials have shown that CAR-T therapy in solid tumors remains unsatisfactory. The main reasons may be related to: 2  1. Difficulties of defining tumor specific targets 2. Tumor antigen heterogeneity 3. Stromal impediment 4. The limited CAR-T cells trafficking to the tumor site 5. Immunosuppressive microenvironment of solid tumor In addition, the safety of CAR-T cell therapy has always been a concern, as it is often associated with serious side effects such as cytokine release syndrome, neurotoxicity (Immune Effector Cell-Associated Neurotoxicity Syndrome) and on-target/off-tumour toxicity (hypogammaglobulinemia). To address these issues, researchers are using dynamic monitoring of CAR-T cells, which would improve understanding of cellular behaviour in vivo, potentially allowing optimisation of infusion timing and dose. It may also help to avoid potentially fatal systemic toxicity. Current monitoring in CAR-T cell studies has mainly focused on disease response assessment, on CAR- T cell persistence, expansion, and effector function, and on serum cytokine and immune marker levels. In solid tumors, the disease response assessment usually depends on the tumor size and morphological change detection by computed tomography (CT) and/or magnetic resonance imaging (MRI), which can provide information about the tumor burden, but the spatial information of infused T cells is undetectable. Therefore, knowing the extent of lymphocyte accumulation at the tumor site is a good biomarker for therapy response. A. Direct labeling approach → Cells can be labelled directly ex vivo with a suitable radiotracer for in vivo imaging B. Reporter imaging strategy → Inserting reporter genes in the immune cells that program proteins which radiopharmaceuticals can specifically target. Direct ex vivo labelling of immune cells involves labelling the cells with an agent that remains trapped within the cells, followed by injection of the cells into the patient. [111In]Inoxine, [89Zr]Zr-oxine and [18F]FDG have been used for this purpose. This technique is widely used in clinical practice because it does not require modification of the cells. There are two main problems with this technique: - The tracer is diluted with each cell division, reducing the measurable signal per cell. - The efflux of the loaded radiotracer reduces the measurable signal and increases the background radiation, resulting in higher levels of administered activity and increased radiation exposure to the patient. Indirect labelling approaches are techniques based on the use of report genes. A reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. This means that report gene imaging constists of introducing genes in a cell of interest under the control of a given promotor. These genes are transduced in specific receptors, proteins or enzymes, that are then targeted after injection of a radio-labelled agent. The advantage of this technique is that the fate of the cells or their progeny can be followed until they die. The method is associated with less cytotoxicity than direct ex vivo labelling. However, care must be taken to ensure that gene editing does not lead to loss of cell function and to avoid immunogenicity. It is also more expensive than direct labelling and requires specialized equipment and training. 3 Several key issues require the most attention: safety, imaging sensitivity and specificity, selection of an appropriate reporter system, and a protocol designed to address real clinical problems. The inducible T cell costimulatory receptor (ICOS) is upregulated during T cell activation and has thus been identified as a potential biomarker for CAR T cell activation. ICOS is not constitutively expressed on resting T cells and is activated upon antigen recognition, with higher expression in CAR T cells than in non-CAR T cells. ICOS could potentially be considered as an exclusive biomarker for activated T cells. Recently, an ICOS targeting tool (89Zr-DFO-ICOS mAb PET) was developed to visualise CAR-T cell activation and predict therapeutic response. The ICOS antibody was evaluated in a murine model of B-cell lymphoma, 5 days after infusion of CD-19-specific T cells. A significantly higher PET signal was detected in the bone marrow of mice treated with CD19 CAR T cells, reflecting their distribution and activation. IMMUNO-PET Immuno-positron emission tomography (immunoPET or iPET) exquisitely fuses the extraordinary targeting specificity of mAb and the superior sensitivity and resolution of PET. This technique is used to assess the progress of CAR-T therapy. For example, specific uptake of tracer seen in the bones suggesting the presence of CAR-T cells infiltrating B-cell Lymphoma (pro). Neverthelss, the use of full-length antibody in this strategy leads to no specific uptake in highly vascularized organs such as heart, spleen, liver. Exhaustion Markers: checkpoint proteins The expression of immune checkpoint proteins on tumour-infiltrating lymphocytes and CAR-T cells is a strong indication of immune tolerance and exhaustion, and a hallmark of treatment failure! Monitoring the expression of immune checkpoint proteins on therapeutic T-cells and in the tumor microenvironment is another potential strategy able to indirect monitoring the long- term efficacy. PRO AND CONS of cell labelling methods - PRO The ability to follow the biodistribution of CAR-T cells in vivo provides important information on whether target engagement has been successful and how the cell uptake changes longitudinally. These data could help predict and stratify which patients will respond to therapy as part of a personalized treatment and could also be used to detect early response to therapy before changes in tumor size are apparent. Cell labelling methods have a wide range of applications in addition to their use in oncology, and these approaches could be of great value for labelling stem cells or other cell therapies in neurological and autoimmune diseases, as well as for studying infectious diseases - CONS The imaging of cell therapies is a relatively new area and a regulatory framework for more routine imaging studies remains to be defined. Most studies to date have involved small numbers of patients from a single institution. Further multicenter studies are needed to provide evidence for both regulators and clinicians. In the future, if cell labelling can be shown to better stratify expensive cell therapies, then the imaging costs can be defrayed by reducing the use of ineffective treatments or replacing them with more effective therapies at earlier timepoints. This will provide evidence to deliver a change in clinical practice, and education and training of both the imaging and oncological communities will facilitate this 4  ADVANCED THERAPEUTIC TECHNOLOGY Lecture 8 (28.11.2024) One nanometer can be expressed in scientific notation as 1x10-9 m, and it is 1/1000 of a micrometer. Quantum dots are small particles that can be measured with a nanometer scale. They are NANOPARTICLES so tiny that quantum effects determine their characteristics. They can be defined as crystals that often consists of just a few thousand atoms. In terms of size, they have the same relationship to a football as a football has to Earth size. Quantum dots have given us new opportunities to create colored lights. Indeed, Quantum dots are now found in commercial products and used across many scientific disciplines, from physics and chemistry to medicine. Since the Greco-Roman period, organic hair dyes obtained from plants such as henna have been used. A consequence of these practices consists of synthesizing lead sulfide nanocrystals to dye hair. With a size of about 5 nm, their appearance is quite similar to PbS quantum dots synthesized by recent materials science techniques. Quantum effects arise when particles shrink. When particles are few nanometers in diameter, the space available for electron wave is less. This affects the particle’s optical properties. Quantum dots absorb light and then emit it on a certain wavelength, and its color depends on the size of the particle. Quantum dots fluorescence properties can be used for research purposes. They are: ultra-high SENSITIVITY (high quantum yield: even a small amount of energy excites these particles to emit light). ultra-high DEFINITION (peaks are narrow (in the picture ↑), so there’s a sharp fluorescence wavelength). ultra-LONG LIFE (stable material). easy FLUORESCENCE CONTROL (size-dependent control). MULTI COLOR imaging (one light source may stimulate different quantum dots). Compared to organic fluorescent probes or proteins such as GFP, the fluorescence intensity of QDs exhibit 10 to 100 times BRIGHTER and the fluorescence STABILITY against photobleaching exhibit 100 to 1000 times higher. If we compare the spectrum of emission of Rhodamine (TRITC) and of a Quantum Dot, we can immediately notice that the quantum dot peak is narrower, so it emits in an extremely specific wavelength, but its main advantage depends on autofluorescence. Indeed, the emission and the excitation have a quite different wavelength, so autofluorescence, that is a general property of the tissue, is not included in the emission peak. 1 PHOTOBLEACHING is a process by which an organic dye or any fluorophore permanently loses its ability to fluoresce due to photochemical alterations. Even in this case, Rhodamine loses its fluorescence in 10 minutes from staining, whereas quantum dots have a longer fluorescence maintenance. Due to their unique characteristics, QDs are vastly useful as a fluorescence label. A number of studies have used QDs as a cell LABELING AGENT, to track cells, intracellular compounds, extracellular vesicles. Moreover, quantum dots can be used for in vivo and ex vivo fluorescence imaging. Many studies have been performed in mice but also in brain for in vivo imaging. In addition to fixed structural imaging, QDs could also be used as probes to study real-time cellular processes. Qdot(QD655) in combination with Halo Tag-technology was used to study the dynamics of the cytosolic myosin motor protein. QDs were conjugated with a Halo-Tag ligand and electroporated into cells, where Halo-Tag ligand-QDs found the protein of interest (myosin) that was fused with the Halo-Tag protein. In this way, QDs were able to indirectly bind to myosin and function as a probe to study its intracellular movements and interactions. Using this technology, the authors were able to observe myosin’s movement along with the actin filament. Quantum dot technique can be applied also for in vivo CANCER DETECTION. Quantum dots with aspartic acid ligands were injected intravenously. QDs-Asp was able to cross the blood–brain barrier and preferably label brain cancer glioma cells. Another attempt was to detect cancer cells (with the recognition of receptors only expressed by them) during surgery or to analyze if tumor margins were correctly cut. QDs’ fluorescence will allow clearly MAP the drug distribution process of QD-derived delivery complexes. The ability of QDs to stay intact when circulating in the body is essential to ensure that QD-derived drug delivery systems are stable until reaching the target site. QDs are easily taken up by various mammalian cell lines and are targeted to different cellular organelles, including the lysosome. This allows the use of the acidic environment in acidic organelles to dissociate therapeutic drugs from the delivery vehicle. Cancer tissues, which are usually more acidic than other normal tissues, can be specifically targeted by these compounds to release the drug. For example, Doxorubicin was loaded on Quantum dots doped with aptamers, which are able to recognize specifically PSMA protein in prostate cancer, to mark with QDs fluorescence the release of Doxorubicin from delivery complex. Quantum dots are nanoparticles with exceptional photobleaching-resistant fluorescence properties, and they have unique optical characteristics are combined with stable physical properties. 2 Nowadays, in clinical applications and trials, they are still not used because they are not easy to degrade once in our body. In diagnosis, QDs can be used to measure low Troponin levels for patients with suspecting heart attack, thanks to the high sensitivity of these particles. Although imaging is the main application of QDs, they can find other applications such as: 1. PHOTOSENSITIZERS (PS) for photodynamic therapy (PDT) 2. PHOTOTHERMAL AGENTS (PA) for photothermal therapy (PTT). PHOTODYNAMIC THERAPY is based on photosensitizer accumulation in the tumor, before being excited by specific wavelength to produce REACTIVE OXYGEN SPECIES in presence of oxygen. Thanks to QDs, the electrons are excited, but they are not able to come back to the ground state. When the electron stays into an intermediate state, it can act directly in the host’s tissue to induce, through ROS formation, cell death. The PHOTOTHERMAL THERAPY is based on an increase in local temperature after irradiation, preferably in the NIR range, which lead to cell death. The use of quantum dots alone has a minimal impact on cancer cells. Instead, when QDs are paired or with chemotherapy or with photothermal/photodynamic therapy, the damage is strong and the viability is decreased, but by pairing these three together, the damage is higher, cells have a very low viability, and even large aggregates are detached. QDs are at most used as DELIVERY AGENTS. They can be also encapsulated in NANOLIPOSOMES: they can express on their membrane a receptor (like PD-1), so the spheres can be injected into the patient to have targeted delivery. Ultrasound can be used on the nanoliposomes to destroy their shell and release the compound in cancer cells, so once they are exposed to a Xenon lamp (for photodynamic therapy), they can kill cancer cells (with the mechanisms previously described). However, they have many drawbacks: - APCs and QDs can interact, reflecting the QDs’ effects on the INNATE IMMUNE SYSTEM. QDs interfere with the maturation of DCs by regulating their differentiation into activator DCs or tolerogenic DCs, and subsets of T-cells and cytokines may be detected because of the immune response induced by QDs. - Endosomes and lysosomes induce cell damage by liberating HEAVY METALS from quantum dots (metal- free compounds or carbon compounds are preferred for the fabrication of QDs to reduce the potential toxicity and promote their safe application). - Pharmacokinetics and biodistribution profiles (for clinical imaging) - Significantly LONGER BLOOD CIRCULATION times than small contrast agents (hours vs minutes) - Long-term ACCUMULATIONS in phagocyte-rich and highly perfused tissues, such as the liver, spleen, lymph nodes, kidneys, and lungs, rising toxicological concerns and compromising (potential) follow-up imaging. 3