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The luminescent-spectral properties of molecules of 2,3,9,10,16,17,23,24-octacarboxyphthalocyanine and its zinc complex embedded in nanoporous silicate gel matrices were studied. It was found that H-aggregates of the phthalocyanines are formed at the initial stage of formation of the silicate framework. It was discovered that the monomeric form of the zinc complex in the gel matrix is restored when the silicate material is dried. The reasons for destruction of the aggregates in the restricted volume of the matrix of nanoscale pores are discussed.

Pre-existing neutralizing antibodies (NAb) to adeno-associated virus (AAV) may diminish the efficacy of AAV-based therapies depending on the titer. To support gene therapy studies in pigs, the seroprevalence of NAb to AAV1, 2, 5, 6, 8, and 9 serotypes were assessed in the sera of 3 different strains of pigs consisting of 60 Norsvin Topigs-20 strain, 22 Gottingen minipigs, and 40 Yucatan minipigs. Cell-based NAb assays were developed for various AAV serotypes. The sera were tested for NAb in a Lec-2 cell line for AAV9 vector and in a COS-7 cell line for the other AAV serotypes. In the 60 Topigs-20 strain 2 to 4 years of age, 100% were positive for AAV2 NAb, 45% positive for AAV6 NAb, and ∼20% positive for each of AAV1, 5, 8, and 9 NAb. These data showed that ∼80% of Norsvin Topigs-20 pigs evaluated were seronegative for pre-existing NAb to the AAV1, 5, 8, and 9 serotypes, respectively. In 22 Gottingen minipigs at 5-6 months of age, serum AAV serotype-specific NAb coexisted with that of various other AAV serotypes at 32% to 46% between two serotypes. These results suggested that coexisting NAb resulted either from multiple AAV serotype coinfection or from one (or more) serotypes that can crossreact with other AAV serotypes in some minipigs. Among the 40 Yucatan minipigs, 20 of the minipigs were3 months old and were all negative for NAb against AAV5, 8, and 9, and only one of these 20 pigs was positive to AAV1 and 6. We further determined the titers in those positive pigs and found most Gottingen minipigs had low titer at 1:20, whereas some of Topigs-20 pigs had titers between 1:80 and 1:320, and some of Yucatan pigs had titers between 1:160 and 1:640. These results suggested that the majority of the pigs in the three strains would be amenable to gene therapy study using AAV1, AAV5, AAV8, and AAV9 and that prescreening on circulating AAV antibodies could be helpful before inclusion of pigs into studies.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement. V-domain immunoglobulin suppressor of T cell activation (VISTA) selectively engages P-selectin glycoprotein ligand-1 (PSGL-1) and suppresses T cells at acidic pH similar to those in tumour microenvironments, thereby mediating resistance to anti-tumour immune responses.

The purpose of this work was to investigate the role of the lymphatic system in the pharmacokinetics of etanercept, a fusion protein. Etanercept 1 mg/kg was administered intravenously (IV) and subcutaneously (SC) to thoracic lymph duct-cannulated and sham-operated control rats. Blood and lymph samples were obtained for up to 6 days. Model-based SC bioavailability of etanercept was 65.2% in the control group. In lymph-cannulated rats, etanercept concentration in the lymph was consistently lower than in serum following IV dosing; and the concentration in the lymph was significantly higher than in serum after SC injection. The absorption occurred predominantly through the lymphatic pathway (82.7%), and only 17.3% by direct uptake into the central compartment (blood pathway). Lymphatic cannulation reduced the area under the serum concentration-time curve by 28% in IV group and by 91% in SC group. A mechanistic pharmacokinetic model that combined dual absorption pathways with redistribution of the systemically available protein drug into lymph was developed. The model successfully captured serum and lymph data in all groups simultaneously, and all parameters were estimated with sufficient precision. Lymphatic system was shown to play an essential role in systemic disposition and SC absorption of etanercept.

Acoustic liquid handlers deliver small volumes (nL-µL) of multiple fluid types with accuracy and dynamic viscosity profiling. They are widely used in the pharmaceutical industry with applications extending from high-throughput screening in compound management to gene expression sequencing, genomic and epigenetic assays, and cell-based assays. The capability of the Echo to transfer small volumes of multiple types of fluids could benefit bioanalysis assays by minimization of sample volume and by simplifying dilution procedures by direct dilution. In this study, we evaluated the Labcyte Echo 525 liquid handler for its ability to deliver small volumes of sample preparations in biological matrix (plasma and serum) and to assess the feasibility of integration of the Echo with three types of bioanalytical assay platforms: microplate enzyme-linked immunosorbent assay, Gyrolab immunoassay, and liquid chromatography with tandem mass spectrometry. The results demonstrated acceptable consistency of dispensed plasma samples from multiple lots and species by the Echo. Equivalent assay performance demonstrated between the Echo and manual liquid procedures indicated great potential for the integration of the Echo with the bioanalytical assay, which allows the successful implementation of microsampling strategies in drug discovery and development.

The growing fraction (GF) of tumor has been reported as one of the predictive markers of the efficacy of chemotherapeutics. Therefore, a semi-mechanistic model has been developed that describes tumor growth on the basis of cell cycle, allowing the incorporation of the GF of a tumor in pharmacokinetic/pharmacodynamic (PK/PD) modeling. Efficacy data of anti-glypican 3 (GPC3) antibody drug conjugate (ADC) in a hepatocellular carcinoma (HCC) patient derived xenograft (PDX) model was used for evaluation of this proposed model. Our model was able to describe the kinetics of growth inhibition of HCC PDX models following treatment with anti-GPC3 ADC remarkably well. The estimated tumurostatic concentrations were used in tandem with human PKs translated from cynomolgus monkey for prediction of the efficacious dose. The projected efficacious human dose of anti-GPC3 ADC was in the range 0.20-0.63 mg/kg for the Q3W dosing regimen, with a median dose of 0.50 mg/kg. This publication is the first step in evaluating the applicability of GF in PK/PD modeling of ADCs. The authors are hopeful that incorporation of GF will result in an improved translation of the preclinical efficacy of ADCs to clinical settings and thereby better prediction of the efficacious human dose.

Исследованы спектрально-люминесцентные свойства молекул 2,3,9,10,16,17,23,24-октакарбокси-фталоцианина и его цинкового комплекса, внедренных в нанопористые силикатные гель-матрицы. Установлено, что на начальной стадии формирования силикатного каркаса образуются Н-агрегаты исследуемых фталоцианинов. Обнаружен эффект восстановления мономерной формы цинкового комплекса в гель-матрице в процессе сушки силикатного материала. Обсуждаются причины разрушения агрегатов в ограниченном объеме наноразмерных пор матрицы.

The development of antibody therapeutics relies on animal models that accurately recapitulate disease biology. Syngeneic mouse models are increasingly used with new molecules to capture the biology of complex cancers and disease states, and to provide insight into the role of the immune system. The establishment of syngeneic mouse models requires the ability to generate surrogate mouse counterparts to antibodies designed for humans. In the field of bispecific antibodies, there remains a dearth of technologies available to generate native IgG-like mouse bispecific antibodies. Thus, we engineered a simple co-expression system for one-step purification of intact mouse IgG1 and IgG2a bispecific antibodies from any antibody pair. We demonstrated proof of concept with CD3/CD20 bispecific antibodies, which highlighted both the quality and efficacy of materials generated by this technology.

Ligand-binding assay (LBA) performance depends on quality reagents. Strategic reagent screening and characterization is critical to LBA development, optimization and validation. Application of advanced technologies expedites the reagent screening and assay development process. By evaluating surface plasmon resonance technology that offers high-throughput kinetic information, this article aims to provide perspectives on applying the surface plasmon resonance technology to strategic LBA critical reagent screening and characterization supported by a number of case studies from multiple biotherapeutic programs.

A sorption material based on modified high-moor peat was developed to be used as a humidity control agent for the preventive conservation of museum pieces in the absence of air-conditioning systems. The prolonged full-scale tests of the peat absorbent in the interiors and depositories of the Belarussian State Museum of People’s Architecture and Life confirmed its high efficiency.

The bioanalytical strategy for monoclonal antibody therapeutics, intended for multiple oncology indications, includes multiple integrated measurements of pharmacologically relevant therapeutics from discovery through development. Three ligand binding assays were cohesively developed and validated, as applicable, using the Gyrolab microfluidic system for the measurement of a free monoclonal antibody BMS-986207. Accuracy and precision demonstrate %bias from −6.3 to 4.4%, percent coefficient of variation (%CV) from 2.6 to 9.8%, and total error from 4.2 to 13.4% in the nonclinical assay; %bias from −0.3 to 3.3%, %CV from 3.5 to 18.2%, and total error from 6.1 to 19.7% in the clinical assay; and >97% of the sample meeting incurred sample reanalysis criteria. The clinical assay was validated using singlicate wells after gaining significant data in the early phase studies to support this cost-effective and efficient strategy. Each assay met fit-for-purpose and/or regulated bioanalytical method validation criteria including stability, selectivity, dilutional linearity, carryover, and specificity criteria with no interference from co-administered monoclonal antibody.

Мета роботи – розробка оптимального методу синтезу нових полізаміщених тієнілпіролів і їх дослідження в якості стимуляторів росту зернових культур.Результати та їх обговорення. Шляхом ступінчастих реакцій були розроблені препаративні методики синтезу 4-(5-карбокситіофен-2-іл)-3,5-диметил-1Н-пірол-2-карбонової кислоти, 5-(5-карбокситіофен-2-іл)-2,4-диметил-1Н-пірол-3-карбонової кислоти та етил-4-(4-аміно-5)-етоксикарбоніл(тіофен-2-іл)-3,5-диметил-1Н-пірол-2-карбоксилату. Найбільш перспективною для дослідження рістстимулюючої активності на насінні зернових стала 4-(5-карбокситіофен-2-іл)-3,5-диметил-1Н-пірол-2-карбонова кислота.Експериментальна частина. Були одержані 4-(5-карбокситіофен-2-іл)-3,5-диметил-1Н-пірол-2-карбонова та 5-(5-карбокситіофен-2-іл)-2,4-диметил-1Н-пірол-3-карбонова кислоти, в яких в якості вихідних сполук використовували етил 4-ацетил-3,5-диметил-1Н-пірол-2-карбоксилат та етил 5-ацетил-2,4-диметил-1Н-пірол-3-карбоксилат. Дією реагенту Вільсмайєра-Хаака на останні виділені відповідні піроли з хлоровініл-карбальдегідним фрагментом, пост-циклізація яких з етиловим естером тіогліколевої кислоти та наступнимгідролізом приводить до утворення тієнілпіролдикарбонових кислот. Дослідження фізіологічної активності синтезованих сполук проводили на насінні різних сортів пшениці та ячменю.Висновки. Шляхом послідовних реакцій синтезували невідомі раніше тієнілпіролкарбонов, Цель работы – разработка оптимального метода синтеза новых полизамещенных тиенилпирролов и их исследование в качестве стимуляторов роста зерновых культур.Результаты и их обсуждение. Путем ступенчатых реакций были разработаны препаративные методики синтеза 4-(5-карбокситиофен-2-ил)-3,5-диметил-1H-пиррол-2-карбоновой кислоты, 5-(5-карбокситиофен-2-ил)-2,4-диметил-1Н-пиррол-3-карбоновой кислоты и этил4-(4-амино-5)-этоксикарбонил(тиофен-2-ил)-3,5-диметил-1H-пиррол-2-карбоксилата. Наиболее перспективной для исследования ростстимулирующей активности на семенах зерновых стала 4-(5-карбокситиофен-2-ил)-3,5-диметил-1H-пиррол-2-карбоновая кислота.Экспериментальная часть. Были получены 4-(5-карбокситиофен-2-ил)-3,5-диметил-1H-пиррол-2-карбоновая и 5-(5-карбокситиофен-2-ил)-2,4-диметил-1H-пирол-3-карбоновая кислоты с использованием в качестве исходных соединений этил 4-ацетил-3,5 диметил-1H-пиррол-2-карбоксилат и этил 5-ацетил-2,4-диметил-1Н-пиррол-3-карбоксилат. Действием реагента Вильсмайера-Хаака на последние выделены соответствующие пирролы с хлорвинилкарбальдегидным фрагментом, пост-циклизация которых с этиловым эфиром тиогликолевой кислоты и последующим гидролизом приводит к образованию тиенилпирролдикарбоновых кислот. Исследование физиологической активности синтезированных соединений проводили на семенах различных сортов пшеницы и ячменя.Выводы. Путем последовательных реакций синтезировали неизвестные ранее полизамещенные тиенилпирролкарбоновые кислоты и их эфиры. Выявлены особенности исследования этих соединений в качестве стимуляторов роста растений., Aim. To develop the optimal method for the synthesis of new polysubstituted thienylpyrroles and study them as cereal growth stimulants.Results and discussion. Preparative methods for the synthesis of 4-(5-carboxythiophen-2-yl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid, 5-(5-carboxythiophen-2-yl)-2,4-dimethyl-1H-pyrrol-3-carboxylic acid and ethyl 4-(4-amino-5)-thoxycarbonyl (thiophen-2-yl)-3,5-dimethyl-1H-pyrrole-2-carboxylate have been developed by step reactions. 4-(5-Carboxythiophen-2-yl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid is the most promising for the study of the growth-stimulating activity on grain seeds.Experimental part. Using ethyl 4-acyl-3,5-dimethyl-1H-pyrrole-2-carboxylate and ethyl 5-acetyl-2,4-dimethyl-1H-pyrrole-3-carboxylate as starting compounds 4-(5-carboxythiophen-2-yl)-3,5-dimethyl-1H-pyrrole-2-carboxylic and 5-(5-carboxythiophen-2-yl) 2,4-dimethyl-1H-pyrrol-3-carboxylic acid were obtained. By the action of the Wilsmeier-Haack reagent on the latter the corresponding pyrroles with the chlorvinylcarbaldehyde fragment were isolated; their post-cyclization with ethyl ester of thioglycolic acid and the subsequent hydrolysis leads to the formation of thienylpyroldicarboxylic acids. The study of the physiological activity of the compounds synthesized was performed on seeds of different varieties of wheat and barley.Conclusions. By means of sequential reactions the polysubstituted thienylpyrrolcarboxylic acids previously unknown and their esters have been synthesized. The features of the study of these compounds as plant growthstimulants have been revealed.

Background: The bioanalytical strategy for antibody–drug conjugates (ADC) includes multiple integrated measurements of pharmacologically relevant ADC. Methods & results: Three ligand-binding assays were validated for the measurement of total antibody, active ADC and total ADC. Accuracy and precision demonstrate %bias from -8 to 14%, %CV from 3 to 11% and total error from 3 to 21%, with >98% samples meeting incurred sample reanalysis criteria. Each assay met stability, selectivity, dilutional integrity, carry over and specificity criteria with no interference from associated metabolite/impurity. Given the active ADC assay sensitivity to payload, active ADC was used to assess drug to antibody ratio. Discussion & conclusion: Implementation of a microfluidic automated platform enabled high throughput sample analysis of multiple analytes with minimal sample processing.

Background: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well. Results: For the quantitation of BMS-986089, both accuracy (99–103%) and precision (2.4–12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100–108%) and precision (0.7–18%) that were obtained for the analysis of the surrogate peptide (VVSVLTVLHQDWLNGK). For Myostatin, accuracy (94–103%) and precision (2.4–14.9%) were obtained for the analysis of the surrogate peptide (IPAMVVDR). Conclusion: The developed method was applied to the analysis of samples following dosing of BMS-986089 to mice. This method highlights the potential of LC–MS/MS-based methods to eventually assess in vivo drug–target engagement.