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Production of a novel type of recombinant IL-2 immunocytokine
Bednaříková, Kristýna ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
Interleukin 2 is a glycoprotein that in humans consists of 133 amino acids and is produced by helper T cells to amplify immune responses. IL-2 has many immunostimulatory and immunoregulatory functions and has been shown to activate the cytotoxic function of natural killer cells, T lymphocytes and monocytes, and to promote cytotoxicity of these effector cells. In therapy, when given in higher doses, IL-2 stimulates effector lymphocytes and defends the body against pathogens and cancer cells. When IL-2 is administered in lower doses, it stimulates T regulatory cells that inhibit the immune response, thus maintaining autotolerance. While studies to date have shown that IL-2 is effective in the treatment of malignancies, considerable toxicity has also been observed. In recent years, studies have been published showing that when IL-2 is covalently bound through an oligopeptide linker to antiIL-2 monoclonal antibodies, there is an increase in its biological activity in vivo. Also, this antibody may sterically hinder binding to certain subunits of IL-2 receptor and thus contribute to the selective activation and expansion of natural killer cells and T effector cells, whereas regulatory T cells are not stimulated. The length of the peptide linker plays a key role in the association of the IL-2 with the anti-IL-2...
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Production of IL-2 fusion antibodies and determination of their biological activity
Frantová, Eliška ; Vaněk, Ondřej (advisor) ; Kubíčková, Božena (referee)
Interleukin 2 is a small cytokine with many important immune functions. It is used mainly as a T cell growth factor, but it also acts on other immune cells, especially NK and NK-T cells. IL- 2 at higher doses induces the differentiation and proliferation of the cell population of effector and memory T cells, which are characterized by cytotoxicity and are able to effectively defend the organism against pathogens and / or tumor cells. In contrast, low-dose IL-2 stimulates the Treg population, which suppresses immune responses and helps prevent autoimmune diseases. However, in cancer therapy, stimulation of this cell population is undesirable. Because free IL- 2 is toxic to the body at high doses, strategies have previously been proposed to potentiate the biological effect of IL-2. One of the most promising appears to be the single-stranded recombinant fusion construct, where IL-2 is covalently linked via an oligopeptide linker to an anti-IL-2 monoclonal antibody (mAb). Based on the findings of the studies of IL-2 / anti-IL-2 mAb immunocomplexes, this immunocytokine (IC) could provide significant therapeutic benefits in vivo, as compared to free IL-2, especially very robust strengthening of biological activity, selective stimulation of specific cell populations according to the selected antibody and...
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Production of interleukin 2 in fusion with monoclonal antibody S4B6
Rožová, Dominika ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
Interleukin 2 is a growth factor of T cells as well as other lymphocytes, such as NK, NKT cells, dendritic and mast cells, which ensure its expression and secretion. IL-2 regulates immune cell homeostasis and is used to treat a variety of disorders including cancer and autoimmune diseases. In recent years, several cases of interleukin 2 complexed with anti-IL2 antibody have been shown to exhibit dramatically higher biological activity in vivo. These complexes have selective stimulatory activity for different IL2 receptors on target cell. This work follows up previous unsuccessful attempts to express and purify a sufficient amount of the murine IL2 immunocomplex with the S4B6 antibody linked by a 15 amino acid long glycine-serine linker. In this work, a plasmid containing the secreted fusion immunocomplex mIL2-S4B6 gene was prepared and stably transfected to the HEK293T cell line using piggyBac system. The protein was then isolated by chelation affinity chromatography and purified by gel permeation chromatography.
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Depletion of Treg cells for potentiation of cancer treatment with HPMA copolymer-bound cytostatic drug conjugates"
Dvořáková, Barbora ; Kovář, Marek (advisor) ; Reiniš, Milan (referee)
Tumor diseases are severe problem worldwide with increasing number of patients suffering from various types of malignancies. Many of approved therapeutics cause serious side toxicities. Therefore, there are intensive efforts to improve cancer treatment protocols. The aim of this study was to deplete regulatory T (Treg) cells without affecting other immunocompetent cells playing a positive role in tumor eradication. Treg cells were reported to hamper anti-tumor immunity and promote tumor growth and survival. Thus, their selective elimination could lead to induction of anti-tumor responses and tumor rejection if combined with chemotherapy with selected N-(2- hydroxypropyl)methacrylamide (HPMA) copolymer-bound drug conjugates. Original approach was to deplete of Treg cells without the use of anti-CD25 mAb that has been widely exploited for Treg cell elimination; however, its long-term persistence in circulation together with inhibitory effect on activated effector cells (CD25+ ) are its main disadvantages. Thus, Treg cells were sensitized to cell cycle-specific cytostatic drugs via application of IL-2/anti-IL-2 JES6.1 mAb immunocomplexes that induce vigorous selective proliferation of this cell population. Subsequent application of cell cycle-specific cytostatics showed steep decrease of Treg cell...
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Production of interleukin 2 in fusion with monoclonal antibody S4B6
Rožová, Dominika ; Vaněk, Ondřej (advisor) ; Kavan, Daniel (referee)
Interleukin 2 is a growth factor of T cells as well as other lymphocytes, such as NK, NKT cells, dendritic and mast cells, which ensure its expression and secretion. IL-2 regulates immune cell homeostasis and is used to treat a variety of disorders including cancer and autoimmune diseases. In recent years, several cases of interleukin 2 complexed with anti-IL2 antibody have been shown to exhibit dramatically higher biological activity in vivo. These complexes have selective stimulatory activity for different IL2 receptors on target cell. This work follows up previous unsuccessful attempts to express and purify a sufficient amount of the murine IL2 immunocomplex with the S4B6 antibody linked by a 15 amino acid long glycine-serine linker. In this work, a plasmid containing the secreted fusion immunocomplex mIL2-S4B6 gene was prepared and stably transfected to the HEK293T cell line using piggyBac system. The protein was then isolated by chelation affinity chromatography and purified by gel permeation chromatography.
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Antitumor activity of IL-2 and IL-7 immunocomplexes in combination with αCTLA-4 and αPD-1 mAbs
Hnízdilová, Tereza ; Kovář, Marek (advisor) ; Hájková, Michaela (referee)
Biological activity of IL-2 and IL-7 in vivo is significantly increased when complexed with some of the respective anti-cytokine mAb. Different immune cell subsets can be preferentially stimulated depending on the anti-IL-2 mAb used to complex IL-2. IL-2/anti-IL-2 mAb S4B6 immunocomplexes (IL-2/S4B6) induce preferential expansion of CD122high cells whereas IL-2/anti-IL-2 mAb JES6-1 immunocomplexes (IL-2/JES6-1) highly selectively stimulate CD25high cells in mice. Similarly, IL-7/anti-IL-7 mAb M25 immunocomplexes (IL-7/M25) possess higher stimulatory activity for both naïve and memory CD8+ T cells in vivo in comparison to free IL-7. CTLA-4 and PD-1 molecules are inhibitory receptors which negatively regulate proliferation, survival and effector functions of T cells. Blocking antibodies against these molecules represent promising immunotherapeutic tool for treatment of malignant diseases. We examined possible synergism of IL-2/S4B6 and αCTLA-4 plus αPD-1 mAbs in tumor-bearing mice. We found that the expansion of recently activated CD8+ T cells driven by IL-2/S4B6 was further augmented by αCTLA-4 plus αPD-1 mAbs. However, these two immunotherapeutic approaches did not show synergistic antitumor activity in any mouse tumor model tested. Next, we showed that IL-7/M25 possessed higher biological activity...
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Cytokine/anti-cytokine mAb complexes and their biological activity
Hnízdilová, Tereza ; Kovář, Marek (advisor) ; Grobárová, Valéria (referee)
biologická aktivita některých cytokinů může být paradoxně zvýšena tvorbou komplexu některými svými monoklonálními protilátkami (mAb) rozpoznávajícími tento cytokin. Prodloužení poločasu eliminace z 2 mAb komplexy, neboť v použitém klonu mAb dochází k selektivní stimulaci buď CD25 2/S4B6 komplex) buněk. Díky výrazné stimulaci NK buněk a paměťových CD8 lymfocytů a pouze mírné stimulaci Treg buněk by IL nahradit konvenční IL vysoce selektivní stimulací Treg buněk naopak mohly uplatnit při léčbě autoimunitních onemocnění a transplantacích. Potenciálně klinicky využitelné jsou dále IL stimulaci žírných buněk, IL plazmatickými buňkami či IL proliferaci a přežívání T lymfocytů. Imunokomplexy mohou být tvořeny také cytokinem a jeho 15Rα komplexy představují generaci IL onistů, které mohou být díky stimulaci expanze NK buněk a paměťových CD8 T lymfocytů využity jako Klíčová slova 15Rα komplexy
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Preparation of plasmids for expression of interleukin 2-fusion monoclonal antibody
Slavata, Lukáš ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
Aim of the thesis was optimization of production of monoclonal antibody against interleu-kin 2 (IL 2) as fusion construct with IL 2 by preparation of new expression vectors based on pTW5 plasmid and by replacing the native signal sequence with new one, originally from secreted alkaline phosphatase. This fusion antibody has interesting biological activity with therapeutic potential - prolonging the half-life of IL 2 in blood circulation. (The thesis is written in Czech.) Powered by TCPDF (www.tcpdf.org)
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Immunocomplexes of IL-2 and anti-IL-2 mAbs as a novel class of selective and extremely potent immunostimulators
Tomala, Jakub ; Kovář, Marek (advisor) ; Smetana, Karel (referee) ; Špíšek, Radek (referee)
vi ABSTRACT IL-2 has been used in cancer therapy and also for other applications like treatment of chronic viral infections or as an adjuvant for vaccines. However, treatment with IL-2 is rather difficult due to its severe side effects. These toxicities, associated with high-dose treatment necessary for IL-2 to function, have been found the most limiting factor for IL- 2 applications. Further, particular anti-IL-2 monoclonal antibodies (mAb) can actually increase biological activity of IL-2 rather than block it. Binding of IL-2 to anti-IL-2 mAb creates a superagonistic immunocomplexes which have dramatically higher and selective biological activity in comparison to free IL-2 in vivo. Such approach may finally over- come the difficulties associated with administration of IL-2, thus opening brand new scopes for IL-2 and its application not only in the field of tumor therapy. We have shown that IL-2 immunocomplexes composed of IL-2 and anti-IL-2 mAb S4B6 (IL-2/S4B6) stimulate predominantly cells expressing CD122 and CD132 (dimeric IL-2 receptor), i.e. NK and MP CD8+ T cells, with Treg, T and NKT cells being expanded as well. IL-2/S4B6 are able to drive the expansion of activated naive CD8+ T cells into functional memory-like CD8+ T cells. Moreover, these immunocomplexes exert therapeu- tical potential alone...
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