The immune system protects us from disease documented by the success story of prophylactic vaccines that efficiently prevent infectious diseases. However, most relevant human diseases are caused by a malfunctioning of the immune system, leading to chronic inflammation, including many types of cancer, chronic infections with pathogens, and various autoimmune and also metabolic diseases. While the development of antibodies against an infectious pathogen can be readily detected in the blood of vaccinated individuals or individuals after infection as a quantifiable measure of specific immunity, we have limited diagnostic options for detecting immune responses carried out by T cells. T cells are critical components of the immune response that recognize and kill infected cells and cancer cells but are also involved in the development of chronic inflammation and disease processes. Immune cells that circulate in the blood are readily accessible for analysis, but most immune responses occur in peripheral tissues where immune cells escape detection by conventional diagnostic approaches. Novel diagnostic tools are urgently needed for Precision Medicine to follow immune responses in disease states within tissues and to detect target engagement of immune therapeutic approaches.
So far, biopsies of disease-affected tissues have long been the mainstay of disease diagnosis to guide and monitor personalized therapy in patients. However, the difficulties and risks associated with obtaining tissue biopsies, particularly in difficult-to-reach tissues, have led to the development of alternative strategies. “Liquid biopsy”, as a blood-based diagnostic approach, was developed to reflect changes in peripheral organs without the need for a tissue biopsy. The liquid biopsy approach is used to analyze circulating free DNA (cfDNA) from cancer tissues. Purification of circulating cfDNA and subsequent mutation analysis are already used for in vitro diagnostic approaches. The analysis of cfDNA provides important information on the genetic changes within cancer tissues but fails to deliver information on inflammation and tissue damage.
Characteristics and applications of cfDNA and exosomes.
Exosomes
Exosomes, a class of small vesicles continuously released from all cells of the body, may serve as a most interesting target for in vitro diagnostics of a liquid biopsy. Exosomes (30-150 nm in size) are the smallest type of extracellular vesicle, produced through inward budding of multivesicular bodies, resulting in intra-luminal vesicles, which are released into the extracellular space by exocytosis. Exosome-associated proteins are currently used to isolate EVs from biological fluids such as blood, urine, or cerebral fluid and identify the cellular source of their origin. Examples of such proteins are tetraspanins (CD9, CD63, CD81), HLA molecules, membrane transport and fusion proteins (Rab 5, Rab 11). Exosomes contain DNA, RNA, microRNA, proteins as well as lipids and, therefore, have the potential to be used as “liquid biopsy” (van Niel et al. 2018, Nature Reviews in Molecular Cell Biology). Since exosomes contain DNA, RNA, lipids and proteins from the cell they are released from, they provide the opportunity to gain a more comprehensive view of the functional state of cells also within tissues.
The molecular composition of exosomes is assumed to reflect (patho-) physiological changes of their cells or tissues of origin. Thus, exosomes secreted from virus-infected cells have the potential to aid in the identification of virus-infected organs and cell populations and the changes associated with infection in cells.
Similarly, exosomes released from cancer cells shall mirror the phenotype of parental as well as metastatic cancer cells. Exosomes released from immune cells are supposed to reflect the phenotype and functional properties of immune cells during ongoing immune responses.
therawis develops innovative technologies to use exosomes for diagnosis and treatment monitoring in viral infection, oncology andimmune-mediated diseases. Diagnostic exosomes (DiEx) will add a novel dimension to in vitro diagnostics by gaining detailed information on virus-infected cells, cancer cells and immune cells. The use of novel markers for detecting and isolating exosomes is the basis for a comprehensive analysis of exosomes in the DiEx strategy pursued by therawis.
