Cпособы получения и перспективы применения биспецифичных антител для лечения онкологических заболеваний

Bispecific antibody molecules contain two different antigen-binding centers. Particular interest in bispecific antibodies is due to their therapeutic application. Two preparations of therapeutic bispecific immunoglobulins, approved for use in the US and European countries, are aimed at the treatment of cancer. Studies published in recent years are devoted to various methods of obtaining monoclonal bispecific antibodies, to study their physicochemical properties, biological activity, preclinical and clinical trials. This paper reviews different approaches  to the production of antitumor bispecific immunoglobulins, as well as the prospects for their practical application.Биспецифичными называют молекулы антител, содержащие 2 разных антигенсвязывающих центра. Особый интерес к молекулам биспецифичных антител обусловлен их терапевтическим применением. Два препарата терапевтических биспецифичных иммуноглобулинов, разрешенные к применению в США и странах Европы, направлены на лечение онкологических заболеваний. Работы, опубликованные в последние годы, посвящены различным способам получения моноклональных биспецифичных антител, исследованию их физико-химических свойств, биологической активности, доклиническим и клиническим испытаниям. Настоящий обзор рассматривает различные подходы к получению противоопухолевых биспецифичных иммуноглобулинов, а также перспективы их практического применения

In vitro initial attachment of HIV-1 integrase to viral ends: control of the DNA specific interaction by the oligomerization state

HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity

Клещевой энцефалит: иммунологические показатели возможного перехода острой стадии в хроническое течение болезни

Several autoimmune diseases with chronic clinical course are characterized by detection of DNA autoantibodies in patients’ serum, while there are no such IgGs in healthy donors’ blood or in patients with acute clinical course with no evidence of chronization. Tick-borne encephalitis has not been considered this way. Several strict criteria have been applied to show that the DNase activity is an intrinsic property of IgGs from the sera of TBE patients but not from healthy donors. The relative activity of IgGs has been shown to vary extensively from patient to patient, but most of the preparations (91%) had detectable levels of the DNase activity. Polyclonal DNase IgGs were not active in the presence of EDTA or after a dialysis against EDTA, but could be activated by several externally added metal ions, with the level of activity decreasing in the order Mn2+ + Ca2+ ≥ Mn2+ + Mg2+ ≥ Mn2+ ≥ ≥ Mg2+ + Ca2+ ≥ Ca2+ ≥ Mg2+ > Ca2+, while K+ , Na+ , Ni2+, Zn2+, and Cu2+ did not stimulate DNA hydrolysis. Affinity chromatography on DNA-cellulose separated the DNase IgGs into many subfractions with various affinities for DNA and very different levels of the relative activity. Possible reasons for catalytic diversity of polyclonal human Abzs are discussed.Ряд аутоиммунных заболеваний с хроническим течением характеризуются обнаружением в крови больных ДНКаутоантител, в то время как их не содержит кровь здоровых доноров или пациентов с острым течением заболеваний, незначительным нарушением иммунного статуса, без определенной склонности к переходу в хронический процесс. Клещевой энцефалит (КЭ) не рассматривался с этих позиций. Предварительно для данной работы проведен поиск достаточно точных критериев обнаружения ДНК-активности антител иммуноглобулина (Ig) G из сыворотки крови больных КЭ и здоровых доноров. Показано, что относительная активность антител IgG значительно варьирует у пациентов, но большинство образцов (91%) имели определяемый уровень ДНКазной активности. Поликлональные ДНКазные антитела IgG не активировались в присутствии ЭДТА или после диализа с ЭДТА, но могли активироваться некоторыми добавленными ионами металлов с уровнем активности, уменьшающимся в ряду Mn2+ + Ca2+ ≥ Mn2+ + Mg2+ ≥ Mn2+ ≥ Mg2+ + Ca2+ ≥ Co2+ ≥ Mg2+ > Ca2+, в то время как K+ , Na+ , Ni2+, Zn2+ и Cu2+ не стимулировали гидролиз ДНК. Аффинная хроматография на ДНК-целлюлозе разделила ДНКазные антитела IgG на множество субфракций с различным сродством к ДНК и очень разными уровнями относительной активности. Возможные причины каталитического разнообразия поликлональных человеческих аутоантител обсуждаются

Producing and prospects for the use of bispecific antibodies for the treatment of cancer

Bispecific antibody molecules contain two different antigen-binding centers. Particular interest in bispecific antibodies is due to their therapeutic application. Two preparations of therapeutic bispecific immunoglobulins, approved for use in the US and European countries, are aimed at the treatment of cancer. Studies published in recent years are devoted to various methods of obtaining monoclonal bispecific antibodies, to study their physicochemical properties, biological activity, preclinical and clinical trials. This paper reviews different approaches  to the production of antitumor bispecific immunoglobulins, as well as the prospects for their practical application

CLINICAL AND DIAGNOSTIC SIGNIFICANCE OF DEOXYRIBONUCLEASE ACTIVITY OF POLYCLONAL IgG AND BLOOD SERA IN PATIENTS WITH SPONDYLOARTHRITIS

Abstract. A group of 266 patients with spondyloarthritidis and 69 healthy persons were included in our study. IgG preparations were isolated from blood sera by a combined rivanol/affine chromatography technique. Homogeneity of IgGs was tested by means of SDS-PAGE. Serum samples from patients and healthy persons, and IgG subclasses 1, 2 and 4 were tested for DNAse activity. A method of DNAse activity measurement was based on rivanol capacity to form a clot with DNA. We have found highly significant differences between the levels of DNAse activity associated with IgG preparations and in blood sera from patients with spondyloarthritidis, and healthy donors (p < 0,0001). DNAse activity of IgG and sera in patients with psoriatic arthritis was higher than in patients with reactive arthritis and ankylosing spondylitis (p < 0,0001). Multiple correlations were revealed between DNAse activity of IgG, blood serum, clinical signs of psoriatic arthritis, reactive arthritis, ankylosing spondylitis, and laboratory findings. We have developed novel tests for differential diagnosis between various disorders, e.g., spondyloarthritidis, based on IgG and serum-associated DNAse activity, corresponding to the criteris of useful and very useful diagnostic tests in rheumatology

Comparison of DNA-Hydrolyzing Antibodies from the Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

It was found that high-affinity anti-DNA antibodies were one of the major components of the intrathecal IgG response in multiple sclerosis (MS) patients [Williamson et al., PNAS, 2001]. Recently we have shown that IgGs from the sera of MS patients are active in the hydrolysis of DNA. Here we have shown, for the first time, that average concentration of total proteins (132-fold), total IgGs (194-fold) and anti-DNA antibodies (200-fold) in the sera is significantly higher than that in the cerebrospinal fluid (CSF) of fifteen MS patients. The relative activities of total protein from sera and CSFs varied remarkably from patient to patient. It was surprising that the specific DNase activity of the total protein of CSF reparations were 198- fold higher than the serum ones. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We present first evidence showing that IgGs from CSF not only bind but efficiently hydrolyze DNA and that average specific DNase activity of homogeneous antibodies from CSF is unpredictably ,49-fold higher than that from the sera of the same MS patients. Some possible reasons of these findings are discussed. We suggest that DNase IgGs of CSF may promote important neuropathologic mechanisms in this chronic inflammatory disorder and MS pathogenesis development.© 2014 Parkhomenko et al

Characterization and selectivity of catalytic antibodies from human serum with RNase activity.

IgG purified from sera of several patients with systemic lupus erythematosus and hepatitis B are shown to present RNA hydrolyzing activities that are different from the weak RNase A-type activities found in the sera of healthy donors. Further investigation brings evidence for two intrinsic activities, one observed in low salt conditions and another specifically stimulated by Mg2+ions and distinguishable from human sera RNases. Cleavage of RNA substrates by the latter activity is not sequence-specific but sensitive to both subtle conformational and/or drastic folding changes, as evidenced by comparative analysis of couples of structurally well-studied RNA substrates. These include yeast tRNAAsp and its in vitro transcript and human mitochondrial tRNALys-derived in vitro transcripts. The discovery of catalytic antibodies with RNase activities is a first step towards creation of a new generation of tools for the investigation of RNA structure