Dalji pravci razvoja terapijskih monoklonskih antitela: novi formati i nove funkcije

Monoclonal antibody therapeutics have dramatically changed the landscape of medicine and human health. In 2022, thirty-six years on from the approval of a first monoclonal antibody, about 100 monoclonal antibodies for treatment of cancer and noncancer indications had secured United States Food and Drug Administration approval. The idea that antibodies could serve as therapeutics emerged over century ago, but it became a reality when scientists developed hybridoma technology for antibody generation in the laboratory. To avoid the shortcomings of the earliest therapeutic monoclonal antibodies of murine origin, which tended to be immunogenic in humans, therapeutic antibody constructs that were more human-compatible (chimeric and humanized) have been produced using genetic engineering technology. Finally, development of phage display, the human antibody mouse, and single B cell antibody innovative technologies, have enabled production of fully human therapeutic antibodies. The successful application of full-size monoclonal antibodies over the last decades has motivated the pharmaceutical company to develop various types of antibody formats in order to improve their efficacy and lower adverse effects. By using strategies to miniaturize and multifunctionalize antibody molecules new classes of antibody therapeutics, such as antibody derivatives (e.g., antibody–drug conjugates and immunocytokines), bispecific/multispecific antibodies, antibody fragments, were developed. In the future, with the progress of modern biotechnology, it can be expected that these new-designed antibodies will finally pave the way for successful treatments of various diseases.Otkriće monoklonskih antitela i njihova primena u terapiji je značajno uticala na lečenje i zdravlje ljudi širom sveta. Danas, tri i po decenije nakon što je prvo monoklonsko antitelo odobreno za upotrebu od strane Agencije za hranu i lekove Sjedinjenih Američkih Država, više od 100 ovih terapeutika za lečenje malignih tumora i drugih bolesti se nalazi na tržištu. Iako je ideja da se antitela mogu koristiti kao lekovi stara više od jednog veka, do njene realizacije je došlo tek kada su naučnici uspostavili tehnologiju za dobijanje hibridoma ćelija čime je bila omogućena proizvodnja antitela u laboratorijskim uslovima. S obzirom da su ta prva terapijska antitela bila mišja i samim tim imunogena za čoveka, razvojem tehnologije genetskog inženjeringa omogućeno je konstruisanje antitela koja su više “humana” (tzv. himerna i humanizovana). Konačno, uspostavljanjem inovativnih platformi za prikazivanje antitela ili njihovih fragmenata na fagima (“phage display”), generisanje miševa koji sintetišu humana antitela ili izolaciju gena za antitela iz pojedinačne B ćelije omogućeno je dobijanje potpuno humanih antitela. I pored više nego uspešne primene celih molekula antitela u terapiji, da bi se prevazišla nedovoljna efikasnost nekih od njih i smanjili neželjeni efekti, farmaceutske kompanije su počele da razvijaju različite formate antitela. Korišćenjem strategije smanjenja veličine i/ili povećanja funkcionalnosti molekula antitela razvijena je potpuno nova klasa terapijskih antitela kao što su derivati antitela (npr. konjugati antitela i lekova), bispecifična/multispecifična antitela, fragmenti antitela i drugi. U budućnosti, sa daljim razvojem moderne biotehnologije, može se očekivati da će ta novodizajnirana antitela omogućiti uspešno lečenje mnogih bolesti.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Imunonefelometrija i imunoturbidimetrija - teorijski principi, instrumenti i primena

Nephelometry and turbidimetry are methods based on the phenomenon whereby light, passing through a medium with dispersed particles is attenuated in intensity by scattering. The intensity of the scattered light is measured at right angle (usually, but not necessarily) to the incident light beam (nephelometry) or at a forward angle (turbidimetry). Based on nephelometry and turbidimetry, immunonephelometric and immunoturbidimetric assays for the measurement of proteins have developed and expanded rapidly in recent years, progressively replacing the time-honored gel precipitation techniques. In this study, the theoretical background of light scattering theory, instrumental systems for nephelometry and turbidimetry, the nature and kinetics of the antibody-antigen reaction in fluid, and the application of immunonephelometric and immunoturbidimetric assays for specific proteins in the clinical laboratory are described.Nefelometrija i turbidimetrija su metode koje sa zasnivaju na rasipanju svetlosti: pri prolasku kroz disperzionu sredinu, svetlost se rasipa na česticama disperzione faze. Intenzitet rasute svetlosti može se meriti pod nekim uglom (uobičajeno, ali ne i obavezno, pod pravim uglom), što predstavlja nefelometriju ili u pravcu upadne svetlosti, što predstavlja turbidimetriju. Primenom nefelometrije i turbidimetrije u imunologiji, razvile su se imunonefelometrija i imunoturbidimetrija, kao savremene tehnike za određivanje proteina i peptida, koje su zamenile klasične, dugotrajne, precipitacione tehnike u gelu. U ovom radu objašnjene su teorijske osnove rasipanja svetlosti, prikazane su sheme i delovi instrumenata nefelometra i turbidimetra i opisane su imunonefelometrija i imunoturbidimetrija koje su u širokoj upotrebi u savremenim kliničkim laboratorijama

Role of the End-Point Mediators of Sympathoadrenal and Sympathoneural Stress Axes in the Pathogenesis of Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis

The role of stress effector systems in the initiation and progression of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the most commonly used experimental model of MS, has strongly been suggested. To corroborate this notion, alterations in activity of the sympathoadrenal and sympathoneural axes of sympathoadrenal system (a major communication pathway between the central nervous system and the immune system), mirrored in altered release of their end-point mediators (adrenaline and noradrenaline, respectively), are shown to precede (in MS) and/or occur during development of MS and EAE in response to immune cell activation (in early phase of disease) and disease-related damage of sympathoadrenal system neurons and their projections (in late phase of disease). To add to the complexity, innate immunity cells and T-lymphocytes synthesize noradrenaline that may be implicated in a local autocrine/paracrine self-amplifying feed-forward loop to enhance myeloid-cell synthesis of proinflammatory cytokines and inflammatory injury. Furthermore, experimental manipulations targeting noradrenaline/adrenaline action are shown to influence clinical outcome of EAE, in a disease phase-specific manner. This is partly related to the fact that virtually all types of cells involved in the instigation and progression of autoimmune inflammation and target tissue damage in EAE/MS express functional adrenoceptors. Although catecholamines exert majority of immunomodulatory effects through beta(2)-adrenoceptor, a role for alpha-adrenoceptors in EAE pathogenesis has also been indicated. In this review, we summarize all aforementioned aspects of immunopathogenetic action of catecholamines in EAE/MS as possibly important for designing new strategies targeting their action to prevent/mitigate autoimmune neuroinflammation and tissue damage

Natural killer cells as participants in pathogenesis of rat experimental autoimmune encephalomyelitis (EAE): lessons from research on rats with distinct age and strain

Natural killer (NK) cells, influencing dendritic cell (DC)-mediated CD4+ lymphocyte priming in draining lymph nodes (dLNs) and controlling spinal cord (SC) infiltration with encephalitogenic CD4+T lymphocytes, modulate EAE (multiple sclerosis model). This study examined their putative contribution to age-related differences in EAE development in Dark Agouti (DA) (exhibiting age-related decrease in EAE susceptibility) and Albino Oxford (AO) (becoming susceptible to EAE with aging) rats. Aging increased NK cell number in dLNs from rats of both strains. In AO rats, but not in DA ones, it also increased the numbers of IFN-gamma-producing NK cells (important for DC activation) and activated/matured DCs, thereby increasing activated/matured DC/conventional Foxp3-CD4+ cell ratio and activated CD25+Foxp3-CD4+ cell number. Aging in DA rats diminished activated/matured DC/conventional Foxp3-CD4+ cell ratio and activated Foxp3-CD4+ cell number. However, MBP-stimulated CD4+ cell proliferation did not differ in dLN cell cultures from young and aged AO rats (as more favorable activated/matured DC/Foxp3-CD4+ cell ratio was abrogated by lower intrinsic CD4+ cell proliferative capacity and a greater regulatory CD25+Foxp3+CD4+ lymphocyte frequency), but was lower in those from aged compared with young DA rats. At SC level, aging shifted Foxp3-CD4+/cytotoxic CX3CR1+ NK cell ratio towards the former in AO rats, so it was less favorable in aged AO rats exhibiting prolonged neurological deficit compared with their DA counterparts. The study showed strain and age differences in number of IFN-gamma-producing NK cells in EAE rat dLNs, and suggested that their pathogenetic relevance depends on frequency and/or activity of other cells involved in CD4+ T cell (auto)immune response

Ageing Affects Thymopoiesis and Experimental Autoimmune Encephalomyelitis Development in a Strain-Dependent Manner

INTRODUCTION: Considering significance of mechanisms of central tolerance for development of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), and suppressive influence of circulating proinflammatory cytokines and alterations in brain-thymus communication, characteristic for the central nervous system (CNS) autoimmune diseases, on thymopoiesis, the study interogated putative strain-based thymus-related specificities relevant for the opposite effects of ageing on susceptibility of Dark Agouti (DA) and Albino Oxford (AO) rats to EAE. METHODS: Quantitative and qualitative changes in thymopoiesis including underlying mechanisms were examined using flow cytometry and RT-qPCR quantification of mRNAs for molecules relevant for integrity of stroma and T-cell development, respectively. RESULTS: With ageing, differently from DA rats, in AO rats the surface density of CD90, a negative regulator of selection threshold, on thymocytes undergoing lineage commitment was upregulated (consistent with TGF-β expression downregulation), whereas the generation of natural CD4+CD25+Foxp3+ regulatory T cells (nTregs) was impaired reflecting differences in thymic expression of cytokines supporting their development. Additionally, specifically in old AO rats, in whom EAE development depends on IL-17-producing CD8+ T cells, their thymic differentiation was augmented, reflecting augmented thymic IL-4 expression. In turn, differently from old DA rats developing self-limiting EAE, in age-matched AO rats developing EAE of prolonged duration, EAE development led to impaired generation of nTregs and accumulation of proinflammatory, cytotoxic CD28-CD4+ T cells in the periphery. DISCUSSION: The study indicates that strain differences in age-related changes in the efficacy of central tolerance, in addition to enhanced thymic generation of CD8+ T cells prone to differentiate into IL-17-producing cells, could partly explain the opposite effect of ageing on DA and AO rat susceptibility to EAE induction. Additionally, it suggested that EAE development leading to a less efficient thymic output of CD4+ cells and nTregs in old AO rats than their DA counterparts could contribute to prolonged EAE duration in AO compared with DA rats. CONCLUSION: The study warns to caution when designing therapeutic interventions to enhance thymic activity in genetically diverse populations, e.g., humans, and interpreting their outcomes. Furthermore, it indicates that CNS autoimmune pathology may additionally worsen thymic involution and age-related immune changes

Lajmska borelioza - epidemiologija, klinička slika i terapija

Lyme borreliosis is a multisystem disorder characterized by a wide spectrum of clinical manifestations. In Europe, the main aetiological agents are Borrelia afzelii, Borrelia garinii and B. burgdorferi sensu stricto, but Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have also been reported as rare or potential causes of human disease. In North America, B. burgdorferi sensu stricto is the only human pathogen. The most common clinical manifestation of Lyme borreliosis is a skin lesion, erythema migrans, which may resolve spontaneously without antibiotic treatment. However, the pathogen can spread to other tissues and organs, causing more severe manifestations that can involve a skin, nervous system, joints, and/or heart, requiring antibiotic therapy. The most commonly used antibiotics in the treatment of Lyme borreliosis are: amoxicillin, ceftriaxone, cefuroxime, doxycycline and azithromycin.Lajmska borelioza je multisistemska bolest koju karakteriše širok spektar kliničkih manifestacija. U Evropi je primarno prouzrokovana vrstama B. afzelii, B. garinii i B. burgdorferi sensu stricto, ali Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii se takođe navode kao retki ili potencijalni uzročnici oboljenja kod ljudi. U Severnoj Americi, kao jedini humani patogen, dokazana je vrsta B. burgdorferi sensu stricto. Najčešća klinička manifestacija lajmske borelioze je kožna lezija, erythema migrans, koja može da prođe spontano, bez terapije antibioticima. Međutim, patogen se može širiti do drugih delova kože i organa i tako prouzrokovati teže manifestacije kako na koži, tako i na nervnom sistemu, zglobovima i/ili srcu, pri čemu je terapija antibioticima neophodna. Najčešće korišćeni antibiotici u terapiji lajmske borelioze su: amoksicilin, ceftriakson, cefuroksim, doksiciklin i azitromicin

Miokarditis - značaj eksperimentalnih modela i autoimunskih mehanizama u etiopatogenezi bolesti

Acute myocarditis is a serious disease and a major cause of dilated cardiomyopathy. Patients with myocarditis may present severe arrhytmia , rapidly progressive heart failure or shock. In humans, about half the cases of myocarditis are preceded by an acute viral infection. Autoimmunity is important in myocarditis: in particular, a reaction to cardiac myosin following viral infection may contribute to development of myocarditis. Experimental autoimmune myocarditis (EAM) is used as an animal model of human giant cell myocarditis. EAM is characterized by severe myocardial damage that includes the infiltration of mononuclear cells into the myocardium and the appearance of multinucleated giant cells. Treatment of acute myocarditis in humans remains a major clinical problem. Although myocardial inflammation and autoimmune process are essential for the progression of the disease, most results do not support routine treatment of myocarditis with immunosuppressive drugs.Akutni miokarditis je klinički ozbiljno oboljenje i glavni uzrok dilatacione kardiomiopatije. Kod pacijenata sa miokarditisom može se u kratkom vremenskom periodu razviti aritmija, progresivna srčana insuficijencija i šok. Polovina svih slučajeva miokarditisa kod čoveka javlja se nakon akutne virusne infekcije miokarda. Pored direktnog oštećenja mišićnih ćelija srca virusom i autoimunski odgovor organizma na miozin, koji se oslobađa iz kardiomiocita tokom virusne infekcije, ima važnu ulogu u patogenezi miokarditisa. Eksperimentalni autoimunski miokarditis (EAM) predstavlja model koji odgovara miokarditisu džinovskih ćelija kod ljudi. EAM karakteriše difuzna mononuklearna ćelijska infiltracija, intersticijski edem i masovna nekroza kardiomiocita. U pojedinim infiltratima se zapažaju karakteristične džinovske višejedarne ćelije. Lečenje akutnog miokarditisa i dalje predstavlja značajan klinički problem. Iako su inflamacija i autoimunski odgovor ključni faktori u razvoju i progresiji bolesti, većina kliničkih studija nije potvrdila opravdanost rutinske primene imunosupresivnih lekova u terapiji miokarditisa

Propranolol diminished severity of rat EAE by enhancing immunoregulatory/protective properties of spinal cord microglia

Sympathetic dysfunction is suggested to contribute to development of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) alike. Considering important role of microglia in development/resolution of neuroinflammation, contribution of noradrenaline, the key sympathetic end-point mediator, in modulation of microglial phenotypic and functional properties in rat EAE model was examined. The study showed that noradrenaline acting in neurocrine and autocrine/paracrine way might influence microglia during EAE. Propranolol treatment over the effector phase moderated EAE course. This was associated with the increased proportion of microglia expressing CX3CR1, the key molecule in their immunomodulatory/neuroprotective action, and up regulation of CX3CR1 downstream Nrf2 gene. This correlated with the increased heme oxygenase-1 (HO-1) expression and phagocytic capacity of microglia, and their phenotypic changes mirrored in increased proportion of CD163- and CD83-expressing cells. The enhanced HO-1 expression was linked with the decreased proportion of microglial cells expressing IL-1 beta and IL-23, and possibly IL-6, followed by increased proportion of IL-10 expressing microglia, and downregulated MCP-1/CCL2 expression. Consistently, spinal cord infiltration with blood-borne myeloid and CD4 + T cells, as well as CD4 + T-cell reactivation/proliferation and differentiation into highly pathogenic IL-17 + cells co-producing IFN-gamma and GM-CSF were decreased in propranolol-treated rats compared with saline-injected controls. The in vitro investigations of the effects of noradrenaline on microglia showed that noradrenaline through beta-adrenoceptor may influence Nrf2 expression also via CX3CR1-independent route. The study suggests beta-adrenoceptor-mediated neuroinflammation-promoting role of noradrenaline in EAE via modulation of microglial Nrf2 expression, and thereby forms the basis for further translational pharmacological research to improve multiple sclerosis therapy

Propranolol reduced severity of EAE by increasing the expression Nrf2 in microglia

Sympathetic dysfunction was proposed to participate in development of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). This may be linked with findings indicating that noradrenaline, the key sympathetic end-point mediator, through β adrenoceptor exerts immunomodulatory action. Considering importance of the target tissue for the clinical outcome of EAE, the study investigated the effects of propranolol, a non-selective β adrenoceptor blocker, on the disease severity in Dark Agouti rats. Administration of propranolol over the effector phase of EAE substantially moderated neurological symptoms of the disease. This correlated with the increased proportion of spinal cord microglia expressing CX3CR1, the crucial neuroinflammation-limiting molecule, and upregulated expression of Nrf2, the key CX3CR1 downstream target gene. Additionally, in spinal cord of propranolol-administered rats the expression of heme-oxigenase 1, Nrf2 target gene, was upregulated. Consequently, microglia from propranolol-administered rats, exhibited increased proportion of IL-10–expressing cells, but decreased those of IL-1β– and IL-23–expressing ones. Propranolol also downregulated the IL-6 and MCP-1/CCL2 expression in spinal cord. Furthermore, propranolol affecting CXCR1/Nrf2 signaling pathway enhanced microglial phagocytic/endocytic capacity and surface expression of anti-inflammatory CD163/CD83 markers. Results from in vitro pharmacological study examining influence of noradrenaline/propranolol on functional properties of microglia showed that microglia synthesize noradrenaline, which, in turn, through β-adrenoceptor, downregulated their Nrf2 expression, in a CX3CR1-independent manner. In accordance with microglial shift towards a more anti-inflammatory profile, in spinal cord of propranolol administered rats was found: i) decreased infiltration with blood-borne myeloid and CD4+ T cells, and ii) reduced CD4+ T-cell reactivation/proliferation and differentiation into highly pathogenic IL-17+ cells co-producing IFN-γ and GM-CSF. The study suggests a neuroinflammation-promoting role for central noradrenaline in EAE, via β-adrenoceptor– mediated modulation of microglial Nrf2 expression. Thus, it points out to a putative target for future translational pharmacological research to optimize multiple sclerosis therapy. Funding: MPNTR RS (grant number 175050

Imunološke i molekularne tehnike u laboratorijskoj dijagnostici virusnih infekcija

Diagnostic virology is presently integrated into routine medical practice. The expanded role of diagnostic virology has several explanations. First, the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) epidemic and the success of organ transplantation have greatly increased the pool of patients at risk for serious opportunistic viral infections. Second, an increasing number of antiviral agents are available, and their use often depends on establishing a laboratory-based diagnosis. Third, technologic developments such as monoclonal antibodies and the polymerase chain reaction (PCR) have made rapid viral diagnosis a reality. An important characteristic of modern diagnostic virology is the use of multiple methods for detecting viral infections. Viral culture, detection of viral antigens and nucleic acids, and detection of viral antibodies are all used to diagnose current viral infection. Many of molecular techniques for detecting of viral genomes are in use in routine diagnostic laboratory to aid viral diagnosis. PCR has been used to detect numerous viruses and is increasingly used to assess prognosis, monitor response to treatment, assess progession of infection, and assess of viral resistance to antiviral drugs. The sensitivity and specificity of PCR and other Nucleic Acid Amplification Techniques (NAATs) have made NAATs the diagnostic assay of choice in routine diagnostic laboratories to replace other less sensitive methods of viral detection.Laboratorijska dijagnostika virusnih infekcija danas je integralni deo medicinske prakse. Sve veća uloga virusološke dijagnostike objašnjava se: i) epidemijom sindroma stečene imunodeficijencije (AIDS), kao i napretkom u transplantaciji organa, što je dovelo do značajnog porasta broja pacijenata koji su podložni razvoju oportunističkih virusnih infekcija, ii) sve većim brojem dostupnih antivirusnih lekova, a njihova primena zavisi od laboratorijski potvrđene dijagnoze, i iii) tehnološkim napretkom i razvojem novih tehnika, kao što su produkcija monoklonskih antitela i lančana reakcija polimeraze (PCR), koje su omogućile brzu dijagnostiku virusnih infekcija. Važna karakteristika moderne dijagnostičke virusologije je korišćenje različitih metoda u detekciji virusnih infekcija. Kultura virusa, detekcija virusnih antigena i nukleinskih kiselina, kao i detekcija virusnih antitela, danas se primenjuju za dijagnozu virusnih infekcija. Mnoge molekularne tehnike za detekciju virusnih genoma primenjuju se u rutinskim dijagnostičkim laboratorijama za postavljanje dijagnoze virusnih infekcija. PCR se koristi za detekciju brojnih virusa, a sve više i za određivanje prognoze bolesti, praćenje uspešnosti primenjene terapije, procenu progresije infekcije i određivanje rezistencije virusa na anti-virusne lekove. Osetljivost i specifičnost PCR i drugih tehnika amplifikacije nukleinskih kiselina (NAATs), učinile su da ove tehnike postanu metode izbora u rutinskim dijagnostičkim laboratorijama i da potisnu ostale manje osetljive metode za detekciju virusa