|
|
Cancer Immunotherapy exploiting engineered antibody fragments against prostate-specific membrane antigen
Das, Gargi ; Bařinka, Cyril (advisor) ; Vaněk, Ondřej (referee) ; Ormsby, Tereza (referee)
Prostate cancer (PCa) remains a leading cause of male cancer-related mortality, necessitating thus the development of novel therapeutic approaches as conventional treatments have limited efficacy. Prostate-specific membrane antigen (PSMA) is an established biomarker for both imaging and therapy of PCa, as it is highly upregulated in neoplastic PCa tissues and metastatic castration- resistant prostate cancer. Consequently, immunological targeting of PSMA has gained significant attention as a therapeutic platform for the management of the disease. The thesis is focused on engineering of antibody fragments and fusion proteins derived from the high affinity anti-PSMA 5D3 monoclonal antibody that can be used as immune cell engagers to target and eliminate PSMA-positive cells. To this end, we engineered 5D3 single chain variable fragments (scFv) that were subsequently fused to anti-CD3 scFv and CP33 sequences, creating thus immune cell engagers targeting T-cells (BiTE) and monocytes (5D3-CP33), respectively. The engagers were expressed in insect cells, purified to homogeneity and their biophysical and functional characteristics evaluated using size exclusion chromatography, differential scanning fluorimetry, ELISA and flow cytometry. Ensuing cell-based assays revealed that both BiTE and 5D3-CP33 can...
|
|
|
Predicting local structural properties from antibody sequence
Beňo, Roman ; Příhoda, David (advisor) ; Hoksza, David (referee)
Predicting local structural properties of antibodies at residual level is vital for detecting the presence of post-translational modifications (PTMs), which often induce structural change in the antibody, negatively impact its shelf-life and possibly lead to the loss of the therapeutic potential. In this work, we predict relative solvent accessibility (RSA) of individual residues. This property is, alongside with the type of amino acid in question, the key indicator for presence of methionine oxidations and other types of PTMs. Due to the conservation of the antibody structure, we identified that different classes of prediction methods yield almost interchangeable results - total mean absolute error (MAE) of 5.64 RSA percentage units measured for the best performing machine learning pipeline compared to the 5.96 measured for the best performing statistical pipeline. The significant prediction quality improvement observed within comparison to the random prediction method with MAE of 35.996 may be as well attributed to the sequence conservancy. In CDR regions, RSA values are harder to predict. Although the range of methods and procedures employed throughout this work is by far not able to yield complex structure predictions, it might constitute a modular, high-throughput tool to support one's choices when...
|
|
|
Implementation and optimization of ELISA method
KOPAČKOVÁ, Tereza
Enzyme-Linked ImmunoSorbent Assay or ELISA is a method that has a wide application, especially in immunology. Antigens and antibodies can be detected by the method. The basic principle of the method is the reaction of antigen and antibody and the subsequent formation of an immunocomplex. The immunocomplex is detected by a conjugate that causes a color change of the substrate in the presence of antigen or antibody in the test sample. The ELISA test is a simple method important fot the diagnosis of various diseases or laboratory research. This method is a special type of enzyme immunoassay. The bachelor thesis is focused on the general implementation and optimization of the ELISA method. The aim of the theoretical part of the work is a general introduction to immunological methods. The division of individual types of ELISA tests, including the principles and use in practice, is elaborated in detail. In the practical part, the ELISA method is introduced and optimized using a DYNAREAD photometer for reading 96well microtiter plates and AlaDYN software. In this way, a total of 8 test samples of sera are examined and subsequently the obtained data are processed. The test measurement was performed using the QuatiVac ELISA test kit and human IgG antibodies against SARS-CoV-2 in serum were determined. The peaks of SARS-CoV-2 spike proteins expresses recombinantly in the human cell line HEK 293 were used as antigens. The measurement was performed using a DYNAREAD instrument and data processing was performer using AlaDYN software. 6 calibrators, a positive control, a negative control and then individual serum samples were measured. The measurement results are 5 serum samples in which IgG class anti-SARS-CoV-2 antibodies were detected and 3 samples in which no antibodies were detected.
|
|
|
Úloha osy PD-1/PD-L1 při infekci \kur{Borrelia burgdorferi} u myší
PALOUNKOVÁ, Anna
Borrelia burgdorferi, the causative agent of Lyme disease, induces upregulation of inhibitory immune checkpoint PD-L1 in mice. We studied if the blockade of PD-1/PD-L1 axis by neutralizing antibodies influences the proliferation of T lymphocytes and cytokine milieu in imunological synapsis between murine dendritic cells and T cells in vitro.
|
|
|
Diagnostic of immunohistochemistry in pathology
HRDINOVÁ, Andrea
The bachelor thesis deals with the use of immunohistochemical methods in the diagnosis of neoplasms in pathology. Describes the use of antibody spectrum at the Department of Pathology, Český Krumlov Hospital, a.s. Immunohistochemical methods are nowadays an important method used in pathology for tumor typing and metastasis for the determination of primary tumors. They complement the standard staining methods for basic diagnostics such as Hematoxylin-Eosin and Van-Gieson, which are less specific, these methods mainly serve to differentiate the basic cellular components and tissue components of the preparation. Immunohistochemistry methods are based on the special antibody affinity principle and are divided into direct and indirect methods. For a direct method, an antibody labeled with, for example, an enzyme is used to detect the antigen and, upon binding, induces a color change observed under the microscope. Indirect method (two-stage, three-stage) is a more sensitive method, based on the principle of an unlabeled primary antibody to which a conjugated secondary antibody binds to an enzyme or biotin. In the three-stage, we use the affinity of avidin and biotin to form the avidin-biotin-enzyme complex (eg. peroxidase), which is shown and highlighted. In more detail, the thesis deals with the diagnosis of breast and sentinel lymph node tumors, both in the theoretical and practical part, and the incidence of these tumors since the introduction of the immunohistochemical method at the Department of Pathology of the Český Krumlov Hospital. Last but not least, a case report of individual interesting cases is described.
|
|
|
Current approaches in the development of vaccines against infectious viral diseases
Vargová, Soňa ; Malý, Petr (advisor) ; Osička, Radim (referee)
Vaccination remains one of the most successful biomedical interventions for preventing viral diseases. While early vaccines were developed by attenuating the infectious agent in cell cultures or by inactivation, new delivery platforms are on the rise thanks to the advent of genetic engineering. The COVID-19 pandemic stimulated the rapid adoption and a massive deployment of these platforms. Viral vector vaccines elicit antigen expression within cells and induce a robust cytotoxic T cell response, unlike protein subunit vaccines conferring mainly humoral immunity. mRNA vaccines also deliver the antigen inside the cells while offering more manageable and faster manufacturing possibilities. Unlike DNA-based vaccines, mRNA does not enter the nucleus, and thus, the probability of disrupting gene expression in the recipient cell is diminished. This thesis aims to offer an overview of current approaches in vaccinology and discuss the various platforms in use. The thesis will also present recent advances in the development of prophylactic vaccines against infections with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) and also will focus on a recently proposed strategy for vaccine development based on non-cognate ligands mimicking epitopes recognised by broadly neutralising antibodies...
|
|
|
Diagnostics and management of aloimunization in pregnancy
Kučerová, Rebecca ; Hornová, Markéta (advisor) ; Žižka, Zdeněk (referee)
The bachelor thesis focuses on the issue of maternal alloimmunization in pregnancy and deals with diagnostic and management of the alloimmunization. This complication would occur in approximately 2000 women in the Czech Republic per a year without a treatment. A pregnant woman is at risk if she is Rh(D) negative and the fetus inherits Rh(D) positivity from father. The development of alloimmunization can be prevented by early preventive dosage of anti-D globulin. Despite a systematically organized system of prophylaxis, a small number of pregnant women still experience immunization. Fetuses are at risk of developing anemia in the uterus and than after birth by developing hyperbilirubinemia. In the most severe cases of intrauterine anemia, the solution is intraumbilical transfusion of erythrocytes. The aim of this study was to summarize the outcomes of IUT in retrospect. The quantitative practical part is completed with 2 model study cases of hydrops fetalis. The quantitative part of the research analyzes the results of all IUTs performed in the period from 1991 to 2021 in the Center of Fetal Medicine of the Gynecology and Obstetrics Clinic of the General Hospital and the First Medical Faculty of Charles University in Prague. Using tables and graphs, the data were then analyzed and described in...
|
|
|
Development of an opsonophagocytic assay for the measurement of functional antibody activity against Bordetella pertussis
Brázdilová, Ludmila ; Bumba, Ladislav (advisor) ; Dráber, Peter (referee)
The Gram-negative pathogen bacterium Bordetella pertussis is the infectious agent causing pertussis or whooping cough. The infection is dangerous to infants, often being deadly if untreated. Since whole-cell pertussis vaccines have been replaced by acellular pertussis vaccines, pertussis has become the most prevalent vaccine-preventable disease in developed countries. Therefore, the development of a new generation of pertussis vaccines has become a high priority. Opsonophagocytic assays are one method used to assess the efficacy of new vaccines. The main objective of the thesis is to develop opsonophagocytic killing and uptake assays for the measurement of functional antibody activity against Bordetella pertussis. Neutrophils from mice and humans were isolated by three different methods and used for the assessment of different human and mouse sera in opsonophagocytic killing and uptake assays. Different experimental conditions were tested, including multiplicity of infection and serum dilutions. The opsonophagocytic uptake assay proved to discriminate between naïve and immune sera. Serum from mice vaccinated with the whole-cell pertussis vaccine enhanced opsonophagocytic uptake of B. pertussis cells into neutrophils, while serum from mice immunized with the acellular pertussis vaccine did not....
|
|
|
Antibody response of anseriform birds against avian schistosomes
HAVELKOVÁ, Markéta
Flukes of the genus Trichobilharzia are typical parasites using aquatic gastropods as intermediate hosts and birds as definitive hosts. However, infectious stages in open water are able to penetrate the skin of a mammal, including humans, and thereby induce a hypersensitivity reaction known as swimmer's itch. Infection with neurotropic species T. regenti in ducks induces an increase in level of IgY antibody which recognize certain parasitic antigens. In this work, these parameters in two other relative but visceral species, T. szidati and T. franki, and cross-reactivity of antibodies in serum of infected duck against heterologous parasitic antigen by ELISA and Western blot were characterized. Antibodies of ducks infected with T. szidati showed very low reactivity with all three parasitic antigens in compare to ducks infected with T. franki or T. regenti. The previously identified strongly immunogenic T. regenti antigens have been evaluated as species non-specific for all trichobilharzia species studied, but useful for avian fluke infection in wild individuals diagnosis.
|
|
|
Preparation of fusion ligands and evaluation of their binding to NK cell receptors
Nepokojová, Tereza ; Vaněk, Ondřej (advisor) ; Grantz Šašková, Klára (referee)
Natural killer cells (NK cells) are an important part of innate immunity. On their surface they express a complex group of receptors that use different signalling motifs to activate or inhibit NK cell cytotoxic activity. NK cells are capable to kill aberrant cells (namely, viral, infected, and tumour cells) by using special cytotoxic mechanisms to trigger apoptosis. The activating receptors recognize tumour or stress-induced ligands, e.g., NKG2D receptor recognizes the MICA ligand and NKp30 recognizes the B7-H6 ligand. Therefore for human immune system it is only natural that cancer cells are destroyed by NK cells. The current therapeutic goals in the treatment of cancer are primarily focused on strengthening the body's own natural ability to fight with cancer and one possible way is stimulation of NK cells to win this deadly fight. In addition to NK cells, antibodies are also widely used for the treatment of cancer, as well as other immune-related disorders. Most of them are monoclonal antibodies, but antibody fragments are getting attention and are being tested more and more in recent years. This work describes the preparation of three bifunctional fusion proteins: B7-H6-L-aHER2, MICA-L-aHER2, and aHER2-L-MICA, which contain immunoligands for the activating receptors of NK cell and VHH fragment...
|
guest ::
