Immunogenicity Of Drugs Research Articles

Bioanalogues in the treatment of rheumatoid arthritis: current status of the problem

The article discusses the use of bioanalogues (BAs) in the treatment of rheumatoid arthritis using the example of the comparable efficacy, tolerability and immunogenicity of the original biologic disease-modifying antirheumatic drug (bDMARD) etanercept (ETC) and its BA GP-2015. We discuss the maintenance of the improvement achieved when switching from the original ETC to BA. Recommendations of international experts and preliminary recommendations of the Association of Rheumatologists of Russia on the use of BA are given. The frequency of development and negative consequences of the nocebo effect when switching patients to BA are described. Data from randomized controlled trials and clinical practice on the safety of switching patients from original biologics to BA are presented. The economic benefits of introducing BA into the clinical practice of rheumatologists in Europe and Russian Federation are considered.

Immunogenicity and Potential for Intraocular Inflammation of Intravitreal Anti-VEGF Drugs

BackgroundConcerns of intraocular inflammation associated with intravitreal administration of anti-VEGF drugs have been risen and the exact mechanism is not yet elucidated. ObjectiveTo explore the relationship between immunogenicity and intraocular inflammation in intravitreal anti-VEGF drugs. MethodsThis review examines the immunogenicity of individual intravitreal anti-VEGF drugs and their potential link to intraocular inflammation. ResultsWe suggest that the main cause of intraocular inflammation is the presence of pre-existing and treatment-induced antidrug antibodies, along with considerations related to the molecular structure, which includes the drug's format and size. ConclusionsResearchers and clinicians involved in the advancement of new anti-VEGF drugs should take into consideration the factors related to intraocular inflammation that have been discussed.

Fabrication of An Immunostimulatory Supramolecular Nanomedicine for Potent Cancer Chemoimmunotherapy.

Chemoimmunotherapy can boost strong antitumor immune responses by triggering immunogenic cell death (ICD), which highlights a promising prospect in clinical applications. However, current chemoimmunotherapy shows limited efficacy due to the low delivery efficiency and insufficient immunogenicity of available chemotherapeutic drugs. A supramolecular polymeric nanomedicine (Pt-Tu@NP) is herein reported using cucurbit[7]uril-based host-guest recognition and noncovalent self-assembly. Pt-Tu@NPs have excellent biodistribution and strongly evoke the endoplasmic reticulum stress-mediated ICD of tumor cells, triggering potent antitumor immune responses by promoting dendritic cell (DC) maturation and cytotoxic T cell infiltration. The coordinated butyrate promotes a positive feedback regulation between DCs and CD8+ T cells. Pt-Tu@NPs stimulate immune cold tumors into hot ones, working in synergy with an immune checkpoint blockade to effectively suppress tumor growth and metastasis, which suggests a promising approach for cancer chemoimmunotherapy.

A blinded in vitro analysis of the intrinsic immunogenicity of hepatotoxic drugs: implications for preclinical risk assessment.

In vitro preclinical drug-induced liver injury (DILI) risk assessment relies largely on the use of hepatocytes to measure drug-specific changes in cell function or viability. Unfortunately, this does not provide indications toward the immunogenicity of drugs and/or the likelihood of idiosyncratic reactions in the clinic. This is because the molecular initiating event in immune DILI is an interaction of the drug-derived antigen with MHC proteins and the T-cell receptor. This study utilized immune cells from drug-naïve donors, recently established immune cell coculture systems and blinded compounds with and without DILI liabilities to determine whether these new methods offer an improvement over established assessment methods for the prediction of immune-mediated DILI. Ten blinded test compounds (6 with known DILI liabilities; 4 with lower DILI liabilities) and 5 training compounds, with known T-cell-mediated immune reactions in patients, were investigated. Naïve T-cells were activated with 4/5 of the training compounds (nitroso sulfamethoxazole, vancomycin, Bandrowski's base, and carbamazepine) and clones derived from the priming assays were activated with drug in a dose-dependent manner. The test compounds with DILI liabilities did not stimulate T-cell proliferative responses during dendritic cell-T-cell coculture; however, CD4+ clones displaying reactivity were detected toward 2 compounds (ciprofloxacin and erythromycin) with known liabilities. Drug-responsive T-cells were not detected with the compounds with lower DILI liabilities. This study provides compelling evidence that assessment of intrinsic drug immunogenicity, although complex, can provide valuable information regarding immune liabilities of some compounds prior to clinical studies or when immune reactions are observed in patients.

Levels of antibodies to adalimumab in children with juvenile idiopathic arthritis at different stages of treatment

Background. Juvenile idiopathic arthritis (JIA) is one of the most common rheumatological diseases of childhood. The central place in the problem of JIA belongs to the question of treatment the timeliness and adequacy of which determine the disease prognosis and, in fact, the entire future of the child. Immunobiological therapy can cause stable clinical and laboratory remission, as well as stop the further progression of structural changes, affecting the pathogenetic link of idiopathic arthritis. But the lack of response to therapy or a decrease in its effectiveness remains a fairly common problem and, in many cases, can be caused by the immunogenicity of immunobiological drugs, especially in case of treatment with tumor necrosis factor inhibitors. Aim of the work: to study the level of antibodies to adalimumab in children with juvenile idiopathic arthritis at different stages of treatment for analysis of immunogenicity. Materials and methods. The concentration of antibodies to adalimumab in 80 serum samples from patients with JIA was studied and evaluated, treatment effectiveness and adverse events were analyzed in 56 patients with JIA at different stages of therapy. Two groups were identified. The first one included 24 patients who had at least a 6-month break in adalimumab administration for non-medical reasons during which treatment was continued with methotrexate with periodic intra-articular injection of glucocorticoids. The level of antibodies to adalimumab was evaluated before the break and 1 month after the reinitiation of adalimumab administration. The second group consisted of 32 children who continued adalimumab without a break during treatment. Disease activity was measured using JADAS-27. Antibodies to adalimumab were detected by enzyme-linked immunosorbent assay. Results. During the examination, an elevated level of antibodies to adalimumab was detected in 10 of 24 serum samples (42 %) before non-medical withdrawal in group I. Among the results of group II, elevated levels of antibodies to adalimumab were found in 12 samples, which was 38 %. The correlation analysis revealed direct statistically significant relationships of moderate strength between the level of antibodies to adalimumab and the indicator of inflammatory activity on JADAS-27 (Spearman’s r = 0.39, p < 0.05), as well as between the level of antibodies and disease duration (Spearman’s r = 0.32, p < 0.05). Conclusions. Monitoring serum antibodies to adalimumab is informative for the correct interpretation of treatment effectiveness and the course of the disease with immunobiological treatment, as it may improve understanding of the clinical consequences of continued therapy, help prevent adalimumab immunogenicity, develop follow-up strategies and, as a result, can affect a long-term outcome of treatment for JIA.

Assessing Immunogenicity of Biologic Drugs in Inflammatory Joint Diseases: Progress Towards Personalized Medicine

Biologic drugs have greatly improved treatment outcomes of inflammatory joint diseases, but a substantial proportion of patients either do not respond to treatment or lose response over time. Drug immunogenicity, manifested as the formation of anti-drug antibodies (ADAb), constitute a significant clinical problem. Anti-drug antibodies influence the pharmacokinetics of the drug, are associated with reduced clinical efficacy, and an increased risk of adverse events such as infusion reactions. The prevalence of ADAb differs among drugs and diseases, and the detection of ADAb also depends on the assay format. Most data exist for the tumor necrosis factor-alpha inhibitors infliximab and adalimumab, with a frequency of ADAb that ranges from 10 to 60% across studies. Measurement of ADAb and serum drug concentrations, therapeutic drug monitoring, has been suggested as a strategy to optimize therapy with biologic drugs. Although the recent randomized clinical Norwegian Drug Monitoring (NOR-DRUM) trials show promise towards a personalized medicine prescribing approach by therapeutic drug monitoring, several challenges remain. A plethora of assay formats, with widely differing properties, is currently used for measuring ADAb. Comparing results between different assays and laboratories is difficult, which complicates the development of cut-offs necessary for guidelines and the implementation of ADAb measurements in clinical practice. With the possible exception of infliximab, limited data on clinical relevance and cost effectiveness exist to support therapeutic drug monitoring as a routine clinical strategy to monitor biologic drugs in inflammatory joint diseases. The aim of this review is to provide an overview of the characteristics and prevalence of ADAb, predisposing factors to ADAb formation, commonly used assessment methods, clinical consequences of ADAb, and the potential implications of ADAb assessments for everyday treatment of inflammatory joint diseases.

Third dose of anti-SARS-CoV-2 inactivated vaccine for patients with RA: Focusing on immunogenicity and effects of RA drugs.

ObjectivesTo evaluate the immunogenicity of the third dose of inactivated SARS-CoV-2 vaccine in rheumatoid arthritis (RA) patients and explore the effect of RA drugs on vaccine immunogenicity.MethodsWe recruited RA patients (n = 222) and healthy controls (HC, n = 177) who had been injected with a third dose of inactivated SARS-CoV-2 vaccine, and their neutralizing antibody (NAb) titer levels were assessed.ResultsRA patients and HC were age- and gender-matched, and the mean interval between 3rd vaccination and sampling was comparable. The NAb titers were significantly lower in RA patients after the third immunization compared with HC. The positive rate of NAb in HC group was 90.4%, while that in RA patients was 80.18%, and the difference was significant. Furthermore, comparison of NAb titers between RA treatment subgroups and HC showed that the patients in the conventional synthetic (cs) disease-modifying anti-rheumatic drugs (DMARDs) group exhibited no significant change in NAb titers, while in those receiving the treatment of biological DMARDs (bDMARDs), Janus Kinase (JAK) inhibitors, and prednisone, the NAb titers were significantly lower. Spearman correlation analysis revealed that NAb responses to SARS-CoV-2 in HC did differ significantly according to the interval between 3rd vaccination and sampling, but this finding was not observed in RA patients. In addition, NAb titers were not significantly correlated with RA-related laboratory indicators, including RF-IgA, RF-IgG, RF-IgM, anti-CCP antibody; C-RP; ESR; NEUT% and LYMPH%.ConclusionSerum antibody responses to the third dose of vaccine in RA patients were weaker than HC. Our study will help to evaluate the efficacy and safety of booster vaccination in RA patients and provide further guidance for adjusting vaccination strategies.

A Randomized, Double-Blind, Single-Dose Study Comparing the Biosimilarity of HOT-1010 With Bevacizumab (Avastin®) in Chinese Healthy Male Subjects.

Objective: This study was conducted to compare the pharmacokinetics, safety and immunogenicity of HOT-1010 with bevacizumab (Avastin®) in Chinese healthy male subjects. Methods: A single-center, randomized, double-blind, single-dose, parallel trial was performed in 84 Chinese healthy male subjects who randomly (1:1) received a single intravenous infusion of 1 mg/kg HOT-1010 or Avastin® for 90 min and followed up for 85 days. Serum concentrations of bevacizumab were analyzed by enzyme-linked immunosorbent assay. Primary pharmacokinetic parameters, Cmax, AUC0-t and AUC0-∞, were calculated and evaluated the bioequivalence between HOT-1010 and Avastin®, the safety and immunogenicity of investigational drugs were also assessed. Results: A total of 82 subjects completed the study. The 90% Confidence Intervals for geometric mean ratios of Cmax, AUC0-t and AUC0-∞ were 91.81–103.64%, 85.19–95.39% and 85.04–95.36%, which were all within the bioequivalence margin. Treatment-emergent adverse events were reported in 27 (65.9%) subjects in HOT-1010 group and 23 (56.1%) subjects in Avastin® group. Most TEAEs were mild or moderate. No TEAEs, Serious Adverse Events or deaths leading to discontinuation was reported. Subjects were all tested negative for Anti-drug Antibody. Conclusion: HOT-1010 exhibited the similar pharmacokinetics, safety and immunogenicity profiles of bevacizumab (Avastin®) in Chinese healthy male subjects. Clinical Trial Registration: http://www.chinadrugtrials.org.cn/index.html, CTR20181610.

In-Vitro Approaches to Predict and Study T-Cell Mediated Hypersensitivity to Drugs.

Mitigating the risk of drug hypersensitivity reactions is an important facet of a given pharmaceutical, with poor performance in this area of safety often leading to warnings, restrictions and withdrawals. In the last 50 years, efforts to diagnose, manage, and circumvent these obscure, iatrogenic diseases have resulted in the development of assays at all stages of a drugs lifespan. Indeed, this begins with intelligent lead compound selection/design to minimize the existence of deleterious chemical reactivity through exclusion of ominous structural moieties. Preclinical studies then investigate how compounds interact with biological systems, with emphasis placed on modeling immunological/toxicological liabilities. During clinical use, competent and accurate diagnoses are sought to effectively manage patients with such ailments, and pharmacovigilance datasets can be used for stratification of patient populations in order to optimise safety profiles. Herein, an overview of some of the in-vitro approaches to predict intrinsic immunogenicity of drugs and diagnose culprit drugs in allergic patients after exposure is detailed, with current perspectives and opportunities provided.

DEVELOPMENT AND PRE-CLINICAL STUDIES OF THE RECOMBINANT VACCINE "BETUVAX-COV-2" FOR PREVENTION OF COVID-19

A candidate vaccine containing a corpuscular adjuvant and a recombinant glycosylated RBD domain of the SARS- CoV-2 virus has successfully passed preclinical studies and is approved for clinical trials. In the course of preclinical studies, the safety and high immunogenicity of the developed drug was shown in experiments on animals, including primates.

A Phase 1 randomized study compare the pharmacokinetics, safety and immunogenicity of HLX02 to reference CN- and EU-sourced trastuzumab in healthy subjects

This study evaluated the bioequivalence of China-manufactured biosimilar, HLX02, to reference China (CN)- and European Union (EU)-sourced trastuzumab. This was a two-part Phase 1 study conducted in healthy Chinese males. Part 1 evaluated the safety of different doses of HLX02 (2, 4, 6 or 8mg/kg; intravenous infusion over 90min, n = 3 per group). Part 2, a randomized, double-blind study, investigated the pharmacokinetics (PK), safety and immunogenicity of study drugs (HLX02 [n = 37], CN-trastuzumab [n = 35] or EU-trastuzumab [n = 37] at the dose suggested by Part 1 results). The primary PK endpoint was the area under the serum concentration-time curve from time 0 to infinity (AUCinf). Equivalence was concluded if the 90% confidence interval (CI) for the geometric least squares mean ratio (GLSMR) fell in the equivalence criteria of 0.80-1.25. In Part 1, all doses of HLX02 were well tolerated and 6mg/kg was suggested for Part 2. The GLSMRs and 90% CIs for AUCinf were: 0.950 (0.891-1.013), 0.914 (0.858-0.973) and 0.962 (0.902-1.025) for HLX02 versus CN-trastuzumab, HLX02 versus EU-trastuzumab and CN-trastuzumab versus EU-trastuzumab, respectively. Secondary endpoints comparisons also fell in the equivalence criteria. Treatment-emergent adverse events were reported in 75.7, 86.5 and 70.3% of the subjects in HLX02, CN-trastuzumab, and EU-trastuzumab groups, respectively. No serious adverse events or deaths occurred. No treatment-related anti-drug antibodies were detected. This study demonstrated comparable safety profiles and PK bioequivalence among HLX02, CN-trastuzumab and EU-trastuzumab in healthy Chinese male subjects. ClinicalTrials.gov, NCT02581748, registered at October 19, 2015.

Assessing the effect of genetic markers on drug immunogenicity from a mechanistic model-based approach

BackgroundWith the growth in use of biotherapic drugs in various medical fields, the occurrence of anti-drug antibodies represents nowadays a serious issue. This immune response against a drug can be due either to pre-existing antibodies or to the novel production of antibodies from B-cell clones by a fraction of the exposed subjects. Identifying genetic markers associated with the immunogenicity of biotherapeutic drugs may provide new opportunities for risk stratification before the introduction of the drug. However, real-world investigations should take into account that the population under study is a mixture of pre-immune, immune-reactive and immune-tolerant subjects.MethodIn this work, we propose a novel test for assessing the effect of genetic markers on drug immunogenicity taking into account that the population under study is a mixed one. This test statistic is derived from a novel two-part semiparametric improper survival model which relies on immunological mechanistic considerations.ResultsSimulation results show the good behavior of the proposed statistic as compared to a two-part logrank test. In a study on drug immunogenicity, our results highlighted findings that would have been discarded when considering classical tests.ConclusionWe propose a novel test that can be used for analyzing drug immunogenicity and is easy to implement with standard softwares. This test is also applicable for situations where one wants to test the equality of improper survival distributions of semi-continuous outcomes between two or more independent groups.

Restoration and Enhancement of Immunogenic Cell Death of Cisplatin by Coadministration with Digoxin and Conjugation to HPMA Copolymer.

Complete tumor eradication is the ultimate goal of cancer therapy. However, the majority of anticancer drugs cause nonimmunogenic cell death and only exert on-site anticancer activities. The intrinsic genomic instability of cancer allows for the persistence and later expansion of treatment-resistant clones after surviving a sort of Darwinian selection of chemotherapy. Additional incorporation of immunotherapy, which is robust and individualized could be game-changing. Herein, we report a combination strategy that delivers nonimmunogenic cell death inducer Cisplatin to treat primary tumors and converts the tumor cells into vaccines that spurs a long-lasting immune response against residual tumors to prevent tumor recurrence and metastasis. Cisplatin(IV) prodrug was linked to the N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer (P-Cis) and coadministered with digoxin (Dig), which eventually launched two attacks to cancer cells. First, P-Cis exhibited superior tumor retention and cytotoxicity over free Cisplatin (to inhibit the primary tumor growth). Then, Dig reversed the inability of Cisplatin to trigger calreticulin exposure, and HPMA copolymer-amplified Cisplatin-induced ATP release. These complementary mechanisms induced potent immunogenic cell death that promotes dendritic cell maturation and activates CD8+ T cell responses. In established tumor models, P-Cis + Dig combination completely eradicate tumors with no residual cancer cells remaining. Cancer cells succumbing to P-Cis + Dig could protect syngeneic mice against the subsequent challenge with living cells of the same type and stimulated robust abscopal and antimetastatic effects. Such a strategy might be promising to restore the immunogenicity of nonimmunogenic drugs and generate vaccine-like functions for improved immunochemotherapy.

A Comparative Parallel Study of Pharmacokinetics and Immunogenicity Following Single Intravenous Administration of Bevacizumab Biosimilar RPH-001 (Manufactured by R-Pharm Group, Russia) and Avastin® (Manufactured by F. Hoffmann-La Roche Ltd., Switzerland) in Healthy Male Volunteers

Introduction. Bevacizumab is a monoclonal IgG1 antibody that binds to and inhibits the biologic activity of human vascular endothelial growth factor (VEGF). Bevacizumab is used as a targeted mono- or combination therapy for different solid tumors. Phase I clinical trial was performed to assess pharmacokinetics (PK) and immunogenicity of bevacizumab drugs. For this study 80 healthy male volunteers were recruited and randomized to either Avastin or RPH-001 group.Aim. To assess and compare pharmacokinetics and immunogenicity (safety) following single intravenous administration of Avastin® (manufactured by F. Hoffmann-La Roche Ltd.,Switzerland) and bevacizumab biosimilar RPH-001 (manufactured by R-Pharm Group,Russia).Materials and methods. Bevacizumab quantitation and quasi-quantitative anti-bevacizumab antibodies detection in human blood serum were carried out using photometric ELISA. Two different methods were successfully validated.Results and discussion. Bevacizumab quantitation method was validated for selectivity and specificity, calibration curve, sensitivity, accuracy and precision, minimal required dilution, dilution linearity and stability. The anti-bevacizumab antibodies detection method was validated for cut-point (with normalization factor calculation), selectivity, sensitivity, precision, drug tolerance, dilution linearity, matrix effect (in case of serum hemolysis), and stability. The validated methods were successfully applied to pharmacokinetic and immunogenicity assessment of bevacizumab drugs. Conclusion. The results of the PK-study showed that test and reference bevacizumab drugs were equivalent. Immunogenicity study did not show any evidence of anti-bevacizumab antibodies in blood serum samples.

Proactively Reducing Anti-Drug Antibodies via Immunomodulatory Bioconjugation.

Although PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to alleviate the development of anti-drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen-presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs.

Proactively Reducing Anti‐Drug Antibodies via Immunomodulatory Bioconjugation

AbstractAlthough PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to alleviate the development of anti‐drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen‐presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs.

The Identification of Critical Quality Attributes (CQAs) for the Development of Antibody Drugs

In the development of quality biopharmaceutical drugs, it is the level of knowledge gained, not the volume of data, which provides the basis for science-based submissions and their regulatory evaluation [International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q8 (R2)]. The identification of critical quality attributes (CQAs) is important as the initial step for quality by design (QbD) strategies. The impact of each potential critical attribute (pCQA) is assessed by using a systematic and scientific approach with respect to bioactivity, pharmacokinetics (PK)/pharmacodynamics (PD), immunogenicity, and safety of the biopharmaceutical drugs. The purpose of this section is to introduce a strategy for identifying CQA, and new analytical methods for CQA assessment.

Scaling of the technology of obtaining the hydroxyaluminium tetra-3-phenylthiophthalocyanine liposomal form and studying its mutagenic and immunotoxic properties

The use of liposomes as drug delivery systems today is a recognized approach to improving the effectiveness of treatment. Compared with other nanosized carriers, liposomes are highly biocompatible. However, in the literature, there are a number of data on the immunogenicity of some liposomal drugs. Hypersensitivity reactions of moderate to severe severity were noted in some patients with intravenous administration of liposomes Doxil®, Ambisome®, DaunoХome®. The technology of production of liposomes is a laborious process and obtaining liposomal forms on an industrial scale is problematic for researchers and manufacturers around the world. In connection with this, studies on the scaling up of technology for obtaining liposomes and studying the specific toxicity of liposomal preparations are relevant. Objective – evaluation of the quality of prototypes of liposomal dosage form based on tetra-3-phenylthiophthalocyanine hydroxyaluminum obtained by scaled technology in comparison with samples produced in laboratory conditions, as well as studying their mutagenic and immunotoxic properties. Object of the study – tetra-3-phenylthiophthalocyanine hydroxyaluminum liposomal, lyophilizate for the preparation of injection for injection of 1,5 mg, in bottles with a capacity of 20 ml (LLF). Materials and methods. Bengem’s method, extrusion, sterilizing filtration, lyophilization, intercellular contact integrity test (for promoter activity), Ames bacterial test, cytogenetic study of chromosomal aberrations, DNA damage test (DNA comet test), Erne method; hypersensitivity reaction of delayed type, determination of mass and cellness of central and peripheral immunity organs, evaluation of phagocytic activity of peritoneal macrophages. Results. Based on the results of the research, the LLF production technology is scaled, which allows obtaining up to 400 LLF flasks per production cycle. It has been established that LLF at doses of 6 and 24 mg/kg does not cause an increase in the number of chromosomal abnormalities in mice and does not cause DNA damage, in doses of 1,1–22,0 μg/dish LLF does not show mutagenic activity in the Ames bacterial test, in the range concentrations of 0,062–3,1 μg in the test for promoter activity does not separate intercellular contacts. It is determined that in doses of 6 and 12 mg/kg, LLF does not affect humoral and cellular immunity, does not change the mass and cellularity of the central and peripheral organs of the immune system. LLF, 24 hours after administration at doses of 6 and 12 mg/kg, dose-dependently decreases the phagocytic activity of peritoneal macrophages, which is restored after 7 days. Conclusion. The technology of LLF production is scaled. In the doses studied, it was found that LLF does not have the ability to promote the process of carcinogenesis and the lack of influence on the humoral and cellular immune response.

Versatile Bioconjugation Chemistries of ortho-Boronyl Aryl Ketones and Aldehydes.

Biocompatible and bioorthogonal conjugation reactions have proven to be powerful tools in biological research and medicine. While the advent of bioorthogonal conjugation chemistries greatly expands our capacity to interrogate specific biomolecules in situ, biocompatible reactions that target endogenous reactive groups have given rise to a number of covalent drugs as well as a battery of powerful research tools. Despite the impressive progress, limitations do exist with the current conjugation chemistries. For example, most known bioorthogonal conjugations suffer from slow reaction rates and imperfect bioorthogonality. On the other hand, covalent drugs often display high toxicity due to off-target labeling and immunogenicity. These limitations demand continued pursuit of conjugation chemistries with optimal characteristics for biological applications. A spate of papers appearing in recent literature report the conjugation chemistries of 2-formyl and 2-acetyl phenylboronic acids (abbreviated as 2-FPBA and 2-APBA, respectively). These simple reactants are found to undergo fast conjugation with various nucleophiles under physiological conditions, showing great promise for biological applications. The versatile reactivity of 2-FPBA and 2-APBA manifests in dynamic conjugation with endogenous nucleophiles as well as conjugation with designer nucleophiles in a bioorthogonal manner. 2-FPBA/APBA conjugates with amines in biomolecules, such as lysine side chains and aminophospholipids, in a highly dynamic manner to give iminoboronates. In contrast to typical imines, iminoboronates enjoy much improved thermodynamic stability, yet are kinetically labile for hydrolysis due to imine activation bythe boronic acid. Dynamic conjugations as such presenta novel binding mechanism analogous to hydrogen bonding and electrostatic interactions. Implementation of this covalent binding mechanism has yielded reversible covalent probes of prevalent bacterial pathogens. It has also resulted in reversible covalent inhibitors of a therapeutically important protein Mcl-1. Such covalent probes/inhibitors with 2-FPBA/APBA warheads avoid permanent modification of their biological target, potentially able to mitigate off-target labeling and immunogenicity of covalent drugs. The dynamic conjugation of 2-FPBA/APBA has been recently extended to N-terminal cysteines, which can be selectively targeted via formation of a thiazolidino boronate (TzB) complex. The dynamic TzB formation expands the toolbox for site-specific protein labeling and the development of covalent drugs. On the front of bioorthogonal conjugation, 2-FPBA/APBA has been found to conjugate with α-nucleophiles under physiologic conditions with rate constant ( k2) over 1000 M-1 s-1, which overcomes the slow kinetics problems and rekindles the interest of using the conjugation of α-nucleophiles for biological studies. With fast kinetics being a shared feature, this family of conjugation chemistries gives remarkably diverse product structures depending on the choice of nucleophile. Importantly, both dynamic and irreversible conjugations have been developed, which we believe will enable a wide array of applications in biological research. In this Account, we collectively examine this rapidly expanding family of conjugation reactions, seeking to elucidate the unifying principles that would guidefurther development of novel conjugation reactions, as well as their applications in biology.

КОРЕКЦІЯ ВРОДЖЕНОГО ІМУНІТЕТУ ІНТАКТНИХ ТА ЩЕПЛЕНИХ ПРОТИ НЬЮКАСЛСЬКОЇ ХВОРОБИ КУРЧАТ З ВИКОРИСТАННЯМ ПРОБІОТИЧНОГО НАНОМЕТАЛОГЛОБУЛІНОВОГО ПРЕПАРАТУ

The development of effective complex drugs for increasing the viability of young farm animals by stimulating the activity of immunity, studying the biological effects of these drugs on the expression of cytokine genes and the formation of humoral immunity by its markers is an urgent problem. The aim of the research was to determine the effect of the complex probiotic-metal-protein preparation (PNMGP) on the dynamics of innate immunity factors in intact chickens and when they are vaccinated with a live vaccine against Newcastle disease. The research was carried out at the NSC "Institute of Experimental and Clinical Veterinary Medicine". Three groups (n = 15) of clinically healthy chickens were formed. Birds of the 1st and 2nd experimental groups received PNMGP, starting from the 5th day of life for 5 days at a dose of 5 g/head, mixed with feed. The third bird group was control. On the 17th day of the experiment, the bird vaccination of the 2nd and the 3rd experimental groups was conducted with a commercial live vaccine against Newcastle disease from La Sota strain . On the 27th, 37th and 47th days of the experiment, 5 heads from each group were euthanized and blood was collected for clinical, biochemical and molecular genetic studies. It has been established that the feeding of PNMGP causes an increase in the number of leukocytes in the blood by 11.3% - 23.9% and the level of hemoglobin by 9.6% - 49.0% relative to the corresponding indices of the control group chickens. The use of the drug causes an increase in the level of iron availability in the organism's of the experimental birds. In particular, the coefficient of saturation of transferrin by iron during the study period in poultry of group 1 was increased by 28.7% -34.3%, a similar effect was found in chickens of group 2. Under the action of PNMGP there are multidirectional changes in the level of markers of non-specific humoral immunity: in blood serum of experimental birds, the level of total protein increases due to the fraction of globulins and content of circulating immune complexes, the suppressive effect of the live vaccine against Newcastle disease on the humoral link of immunity is reduced - the level of Sm in the birds of the 2nd group was lower than that of the chickens of the 3rd group by 10,0%, 8,6% and 25,0% on the 10th, 20th and 30th days after vaccination, respectively. In chickens of group 1, the expression level of IL-2 gene exceeded the control values by 91.1%, and 88.8% on the 27th and 47th days of the experiment respectively, and on the 37th day - 10.7 times; and IL-17 - by 50.0% and 59.2% on the 27th and 47th days, respectively, on the 37th day - 3.5 times. The intensity of expression of IL-2 and IL-17 genes in chickens of group 2 was maximally increased - 20.7 and 2.4 times, respectively. The data we have obtained show the expressed immune modulating effect of PNMGP on the state of bird innate immunity. This contributes to the enhancement of the synthesis of specific post-vaccine antibodies to the Newcastle disease virus: their level in the birds of the 2nd group exceeded the control group's indices by 14.3% - 25.7% during the research period. The results we obtained, by the determined trends in the development of immune responses when using a complex probiotic nanometal globulin preparation, coincide with the results of other researchers and can be used as a basis for the development and application of means for enhancing innate immunity of animals and immunogenicity of vaccine drugs. Key words: Complex probiotic nanometal globulin preparation, innate immunity, poultry, expression of cytokine genes, vaccination, specific immunity