|
|
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...
|
|
|
Structural studies of metal-dependent hydrolases: Histone Deacetylase 6 and Glutamate Carboxypeptidase II
Shukla, Shivam ; Bařinka, Cyril (advisor) ; Stříšovský, Kvido (referee) ; Kolenko, Petr (referee)
Zinc-dependent hydrolases are a class of metalloenzymes that require zinc ions to catalyse hydrolytic reactions. Structural studies of these enzymes shall provide detailed information about the processing of their natural substrates, domain organization, and overall structural fold. This thesis describes the structural properties of two different metallohydrolases 1) human histone deacetylase 6 (HDAC6) and 2) glutamate carboxypeptidase II (GCPII) by utilizing a different set of biophysical techniques. HDAC6 is a structurally unique multidomain enzyme comprised of unstructured and globular domains. It regulates the plethora of cellular processes by removing an acetyl group from lysine side chains of target proteins. It has been known to deacetylate non-histone substrates such as tubulin, Hsp90, cortactin, and peroxiredoxins. Given its structural complexity, complete structural information of full-length HDAC6 is missing and available information is limited to its globular domains only. Hence, the integrative approach was employed in combining experimental data from several orthogonal biophysical techniques to build an in-solution structural model of HDAC6. The study reports that HDAC6 adopts multiple conformations due to its unstructured regions and exists as an ensemble of conformers in solution....
|
|
|
Characterisation of recombinant mouse glutamate carboxypeptidase III
Janoušková, Karolína ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Glutamate carboxypeptidase II (GCPII, PSMA, NAALADase) is transmembrane metalopeptidase and due to cleavage of substrates β-citryl-L-glutamate (BCG), N-acetyl-L-aspartyl-L-glutamate (NAAG) and polyglutamylated folates (Pte-Glun) is being studied as potential therapeutic target. Enzymes, which could compensate for enzyme activity and functions of GCPII, are thus relevant targets of enzymology as well. One of GCPII's homologs with similar enzyme activity is mouse glutamate carboxypeptidase III (GCPIII, NAALADase II). Enzymatic cleavage has not been determined using recombinant mouse GCPIII yet. It is important to kinetically characterize mouse GCPIII so that we can compare enzyme activity with human ortolog. Then we can find out whether mouse model is comparable with human. Recombinant mouse GCPIII was kinetically characterized. Kinetic parameters (KM, kcat) for recombinant mouse GCPIII were measured for substrates NAAG and BCG using radioactive assay. Experiments with the substrate Pte-Glu2 were analyzed using HPLC method. Although human GCPIII is more effective than mouse ortolog at clearage of NAAG, both enzymes are comparable during hydrolysis of BCG. Those results can contribute to better understanding of the role of GCPIII in the most commonly used animal model.
|
|
|
Antibody derivatives for the detection of human glutamatecarboxypeptidase II
Bělousová, Nikola ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Prostate cancer is one of the most common human malignancies and, consequently it is critical to develop appropriate diagnostic and therapeutic tools. Glutamate carboxypeptidase II (GCPII) is currently being considered one of the most important prostate cancer markers due to its tissue- specific expression. Whereas in healthy prostatic tissue the expression levels of GCPII are low, the transformation into the tumor is associated with the substantial increase of GCPII expression, with the highest levels observed in androgen-independent metastatic tumors. GCPII is thus considered a promising marker for early phase as well as advanced metastatic stages of prostate cancer. Current research is focused on the development of highly sensitive and specific reagents that allow detection of small amounts of GCPII, for example in early stages of cancer. Antibody derivatives are promising molecules for this purpose because they have high affinity and specificity and minimum negative side effects. Protein engineering is a prefered approach for preparation of various antibody molecules that differ in size, binding properties, stability, solubility, and production means. Different types of derivatives are being developed for medical needs such as in vitro diagnosis, therapy, and in vivo imagingSmall molecular...
|
|
|
Cloning, expression and biochemical characterisation of mouse glutamate carboxypeptidase II.
Knedlík, Tomáš ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
English Abstract Glutamate carboxypeptidase II (GCPII) is a membrane metallopeptidase expressed in many human tissues, predominantly in prostate, brain and small intestine. In brain it cleaves the most abundant peptide neurotransmitter N-acetyl-L-aspartyl-α-L-glutamate into N-acetyl-L-aspartate and free L-glutamate. Thus, GCPII participates in glutamate excitotoxicity through the release of free glutamate into the synaptic cleft. Inhibition of this activity has been shown to be neuroprotective in rats. In the human jejunal brush border, GCPII cleaves off terminal glutamate moieties from poly-γ-glutamylated folates, which can be then transported across the intestinal mucosa. The function of GCPII in human prostate is unknown but it is overexpressed in prostate cancer. Therefore, GCPII is an important marker of prostate cancer and its progression.Moreover, it could become a perspective target for treatment of prostate cancer as well as neuronal disorders associated with glutamate excitotoxicity. For the development and testing of novel drugs and therapeutics it is necessary to have an appropriate animal model. Mouse (Mus musculus) is such a model and it is widely used by many experimentators. However, no detailed comparison of mouse and human GCPII orthologs regarding their enzymatic activity, inhibition...
|
|
|
Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Novák, Petr (referee)
Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...
|
|
|
Příprava a charakterisace rekombinantního dermcidinu jako potenciálního proteinového partnera glutamátkarboxypeptidasy II
Tužil, Jan ; Konvalinka, Jan (advisor) ; Pavlíček, Jiří (referee)
A process of forming new blood vessels is necessary for tumour viability and expansion. Without vasculature, tumour stops growing at a size of millimeters. Some tumours, however, undergo an angiogenic switch and start to build up their own vascular architecture. The rate of apoptosis then decreases and the tumour becomes invasive. There are many factors that control the process of physiological angiogenesis. These might or might not relate to tumour tissue as well. Glutamate carboxypeptidase II (GCPII; EC 3.4.17.21) is a type II transmembrane glycoprotein with two known enzymatic activities. GCPII expression is upregulated in prostate cancer and also highly expressed in tumour-associated neovasculature even though none of these enzymatic functions was observed on the endothelium. Although numerous researches suggested that GCPII might serve as a receptor, no natural ligand has been identified yet. Preliminary experiments performed in our laboratory indicated some proteins to be possible natural ligands of GCPII. Therefore, we chose one of them- dermcidin, cloned and expressed this protein in mammalian cells. We investigated its possible interaction with GCPII introducing new detection system utilizing FLAG-tag however, we were not able to approve neither disapprove its interaction in vitro.
|
|
|
Physiological and pathophysiological role of GCPII in the body
Sedlák, František ; Konvalinka, Jan (advisor) ; Klener, Pavel (referee) ; Smetana, Karel (referee)
Glutamate carboxypeptidase II (GCPII) is a metalloprotease responsible for cleaving the neurotransmitter N-acetyl-aspartyl-glutamate in the central nervous system to N-acetyl aspartate and glutamate. At the same time, in the human small intestine, it facilitates folate absorption by cleaving γ-linked glutamate from folyl-poly-γ-glutamate. In humans, GCPII is also expressed in a number of other organs (e.g., kidney and prostate) and tumors, where its physiological function is unknown. In an attempt to characterize the physiological function of the enzyme, we first characterized the commercially available monoclonal antibodies against GCPII. Further, we developed a fully synthetic replacement based on a hydrophilic polymer with bound GCPII inhibitors. We evaluated the suitability of using a murine biomodel to study GCPII function in vivo. We found the difference in GCPII expression profile in mouse and human. We did not observe GCPII in either the mouse prostate or small intestine. To assess physiological and pathophysiological functions of the enzyme we analyzed a GCPII-deficient mouse model. Apart from the observation of enlarged seminal vesicles in older males, we did not detect any other obvious phenotype. Similarly, we confirmed that GCPII cannot cleave amyloid peptides (Aβ1-40 and Aβ1-42)....
|
|
|
Structure-assisted development of a continuous carboxypeptidase assay
Rakhimbekova, Anastasia ; Bařinka, Cyril (advisor) ; Bouřa, Evžen (referee)
Glutamate carboxypeptidase II (GCPII) is a zinc-dependent carboxypeptidase with high expression levels in prostate carcinoma. As the enzyme represents a validated target for cancer therapy and imaging, the development of new GCPII-specific ligands is still a focus of an active academic and industrial research. However, existing assays to screen inhibitor libraries and determine inhibitor efficacy are suboptimal at best. This thesis is aimed at the development of small internally quenched probes that could be used for continuous measurement of the GCPII enzymatic activity. These probes are derived from natural GCPII substrates and consist of a fluorophore/quencher pair connected by a GCPII-hydrolysable linker. I first characterized biophysical properties of the probes and then determined kinetic parameters of their hydrolysis by GCPII. The optimized activity assay was then used to determine inhibition constants of several GCPII-specific inhibitors. Finally, complexes between the inactive enzyme and several probes were co-crystallized and one of the complexes refined and analyzed. Our data show that the probes are involved in non-covalent interactions with the same amino acid residues of the enzyme's active site as natural substrates. The developed assay could be optimized for high-throughput...
|
|
|
Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica
Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...
|
guest ::
