99mTc-HYNIC/Cy7-Fab(Bevacizumab): su empleo como agente de imagen en mieloma múltiple / 99mTc-HYNIC/Cy7-Fab(Bevacizumab): its use as an imaging agent in Multiple Myeloma / 99mTc-HYNIC/Cy7-Fab (Bevacizumab): seu uso como agente de imagem em mieloma múltiplo

Introducción: el mieloma múltiple es un trastorno hematológico maligno y el segundo cáncer de la sangre más frecuente. El proceso de la angiogénesis tumoral es fundamental para el crecimiento y metástasis de muchos tipos de tumores, incluido en mieloma múltiple. Se sabe que la sobreexpresión del factor de crecimiento endothelial vascular se encuentra asociado a un mal pronóstico en esta patología, representando un blanco clave para la terapia anti-angiogénica en mieloma múltiple. El anticuerpo monoclonal Bevacizumab es capaz de unirse con gran afinidad al factor de crecimiento endothelial vascular bloqueando su acción. Objetivo: evaluar el Fab(Bevacizumab) marcado con 99mTc o Cy7 como potenciales agentes de imagen moleculares de la expresión de factor de crecimiento endothelial vascular en mieloma múltiple. Material y métodos: la expresión de factor de crecimiento endothelial vascular fue analizada mediante citometría de flujo en la línea celular huaman de mieloma múltiple, la MM1S. Fab(Bevacizumab) fue producido mediante digestión de Bevacizumab con papaína, conjugado a NHS-HYNIC-Tfa y radiomarcado con 99mTc. Se realizaron estudios de biodistribución y de tomografía computarizada por emisión del fotón simple. A su vez, Fab(Bevacizumab) fue marcado con Cy7 para obtener imágenes de fluorescencia in vivo hasta 96 horas. Resultados: el análisis por citometría de flujo en la línea celular MM1S reveló que la expresión de factor de crecimiento endothelial vascular es predominantemente intracelular. Los estudios de biodistribución y SPECT/CT del complejo 99mTc-HYNIC-Fab(Bevacizumab) mostraron una rápida eliminación sanguínea y una significativa captación a nivel renal y tumoral. Las imágenes por fluorescencia empleando Cy7-Fab(Bevacizumab) permitieron la visualización tumoral hasta 96 h p.i. Conclusiones: logramos visualizar la expresión de factor de crecimiento endothelial vascular in vivo en mieloma múltiple mediante el empleo del fragmento Fab del anticuerpo anti-VEGF (Bevacizumab) marcado con 99mTc y Cy7. Estos nuevos agentes de imagen molecular podrían ser empleados potencialmente en el ámbito clínico para la estadificación y el seguimiento de pacientes con mieloma múltiple, mediante la visualización radioactiva in vivo de la expresión de factor de crecimiento endothelial vascular en todo el cuerpo. La imagen óptica de estos trazadores mejoraría el muestreo tumoral y podría guiar la extirpación quirúrgica.

Introduction: Multiple myeloma is a hematologic malignancy and the second most common blood cancer. The process of tumor angiogenesis is central to the growth and metastasis of many types of tumors, including multiple myeloma. Overexpression of vascular endothelial growth factor is known to be associated with poor prognosis in this pathology, representing a key target for anti-angiogenic therapy in multiple myeloma. The monoclonal antibody Bevacizumab is able to bind with high affinity to vascular endothelial growth factor blocking its action. Objective: to evaluate 99mTc- or Cy7-labeled Fab(Bevacizumab) as potential molecular imaging agents of vascular endothelial growth factor expression in multiple myeloma. Methods: Vascular endothelial growth factor expression was analyzed by flow cytometry in the multiple myeloma huaman cell line, MM1S. Fab(Bevacizumab) was produced by digestion of Bevacizumab with papain, conjugated to NHS-HYNIC-Tfa and radiolabeled with 99mTc. Biodistribution and single photon emission computed tomography studies were performed. In turn, Fab(Bevacizumab) was labeled with Cy7 to obtain in vivo fluorescence images up to 96 hours. Results: Flow cytometry analysis in the MM1S cell line revealed that vascular endothelial growth factor expression is predominantly intracellular. Biodistribution and SPECT/CT studies of the 99mTc-HYNIC-Fab(Bevacizumab) complex showed rapid blood clearance and significant renal and tumor uptake. Fluorescence imaging using Cy7-Fab(Bevacizumab) allowed tumor visualization up to 96 h p.i. Conclusions: we were able to visualize vascular endothelial growth factor expression in vivo in multiple myeloma using the Fab fragment of the anti-VEGF antibody (Bevacizumab) labeled with 99mTc and Cy7. These new molecular imaging agents could potentially be employed in the clinical setting for staging and monitoring of patients with multiple myeloma by in vivo radioactive visualization of vascular endothelial growth factor expression throughout the body. Optical imaging of these tracers would improve tumor sampling and could guide surgical excision.

Introdução: O mieloma múltiplo é uma malignidade hematológica e o segundo câncer de sangue mais comum. O processo de angiogênese tumoral é fundamental para o crescimento e a metástase de muitos tipos de tumores, incluindo o mieloma múltiplo. Sabe-se que a superexpressão do fator de crescimento endotelial vascular está associada a um prognóstico ruim no mieloma múltiplo, representando um alvo importante para a terapia antiangiogênica no mieloma múltiplo. O anticorpo monoclonal Bevacizumab é capaz de se ligar com alta afinidade ao fator de crescimento endotelial vascular e bloquear sua ação. Objetivo: avaliar o Fab(Bevacizumab) marcado com 99mTc ou Cy7 como possíveis agentes de imagem molecular da expressão do fator de crescimento endotelial vascular no mieloma múltiplo. Métodos: A expressão do fator de crescimento endotelial vascular foi analisada por citometria de fluxo na linha celular de mieloma múltiplo MM1S. O Fab(Bevacizumab) foi produzido pela digestão do Bevacizumab com papaína, conjugado com NHS-HYNIC-Tfa e radiomarcado com 99mTc. Foram realizados estudos de biodistribuição e tomografia computadorizada por emissão de fóton único. Por sua vez, o Fab(Bevacizumab) foi marcado com Cy7 para geração de imagens de fluorescência in vivo por até 96 horas. Resultados: A análise de citometria de fluxo na linha celular MM1S revelou que a expressão do fator de crescimento endotelial vascular é predominantemente intracelular. Os estudos de biodistribuição e SPECT/CT do complexo 99mTc-HYNIC-Fab(Bevacizumab) mostraram uma rápida depuração sanguínea e uma captação renal e tumoral significativa. A imagem de fluorescência usando Cy7-Fab(Bevacizumab) permitiu a visualização do tumor até 96 horas p.i. Conclusões: Conseguimos visualizar a expressão do fator de crescimento endotelial vascular in vivo no mieloma múltiplo usando o fragmento Fab do anticorpo anti-VEGF (Bevacizumab) marcado com 99mTc e Cy7. Esses novos agentes de imagem molecular poderiam ser usados no cenário clínico para o estadiamento e o monitoramento de pacientes com mieloma múltiplo, visualizando radioativamente a expressão do fator de crescimento endotelial vascular in vivo em todo o corpo. A geração de imagens ópticas desses traçadores melhoraria a amostragem do tumor e poderia orientar a excisão cirúrgica.

Prognostic Significance of LPCAT1 in Adult Acute Myeloid Leukemia Patients with FAB Subtype M2 / 中国实验血液学杂志

OBJECTIVE@#To study the prognostic value of LPCAT1 in acute myeloid leukemia (AML).@*METHODS@#TaqMan-based reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect relative expression of LPCAT1 in 214 newly diagnosed adult AML patients and 24 normal controls. Survival functions were estimated using the Kaplan-Meier method and were compared by the Log-rank test. A Cox proportional hazard regression model was used to identify prognostic factors.@*RESULTS@#The expression level of LPCAT1 in adult AML was 34.37%(1.83%-392.63%), which was significantly lower than 92.81%(2.60%-325.84%) of normal controls (P<0.001). The prognostic significance of LPCAT1 was evaluated in 171 non-acute promyelocytic leukemia patients with complete clinical information and prognostic data. Survival analysis showed that the expression level of LPCAT1 had no significant effect on the prognosis of the whole cohort. However, in AML patients with FAB subtype M2 (AML-M2), the 2-year relapse-free survival (RFS) rate of patients with low LPCAT1 expression was 35.4%(95%CI: 0.107-0.601), which was significantly lower than 79.2%(95%CI: 0.627-0.957) of patients with high LPCAT1 expression (P=0.012). Multivariate analysis showed that low expression of LPCAT1 was an independent risk factor for RFS of AML-M2 patients (HR=0.355, 95%CI: 0.126-0.966, P=0.049).@*CONCLUSION@#In adult AML patients LPCAT1 shows low expression. Low LPCAT1 expression is an independent risk factor for RFS in M2-AML patients.

Action of Bromelain and Ficin on horse anti Bothrops sp venom Antibodies

Abstract The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)'2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)'2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Streamlined downstream process for efficient and sustainable (Fab)2 antivenom preparation

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)

LC-MS analysis of sintilimab as an anti-PD-1 therapeutic mab for its improved hinge stability study / 药学学报

Monoclonal antibodies (mAbs) have been widely used as therapeutic drugs for treating diseases such as cancers and auto-immune diseases. When using an IgG4 isotype, one of the challenges is the instability of its hinge which is prone to Fab-arm exchange (FAE). The hinge sequence of a wild type IgG4 is -CPSC-, however, a single point mutation S228P from -CPSC- to -CPPC- can effectively diminish FAE, thereby improving hinge stability of the IgG4 molecule. Sintilimab is the fully human anti-PD-1 monoclonal antibody designed and developed for immuno-oncology, in which serine 228 in the hinge was engineered to proline to mitigate FAE. In this study, LC-MS is used to study hinge stability of sintilimab in both in vitro (PBS and human serum) and in vivo (SCID mouse) studies. The studies demonstrate that LC-MS is a fast and simple way to monitor for the occurrence of FAE in vitro and in vivo, and FAE can be eliminated by antibody engineering with a single point mutation.

Generation of human monoclonal antibody Fab fragment to Zika Virus by Phage Display Technology / 中华实验和临床病毒学杂志

Objective@#In this study, phage display technology was used to construct the human anti-Zika virus(ZIKV), phage antibody library and to obtain and express the monoclonal antibody. The aim was to master the preparation and expression of human phage antibody library screening method for highly specific antibodies.@*Methods@#The whole blood samples of Zika patients were collected and the lymphocytes were isolated. The RT-PCR method was used to amplify the antibody light chain and heavy chain Fab gene from lymphocyte Ig mRNA. The pComb3H system was used to construct the gene with genetic diversity Preparation of human anti-ZIKV phage antibody library. The purified antibody library was screened by using the purified ZIKV and the obtained ZIKV E protein antigen.@*Results@#The monoclonal antibody Fab fragment gene was successfully obtained for the ZIKV E protein antigen. The gene can be efficiently expressed in Escherichia coli.@*Conclusions@#According to the sequence analysis, this study showed that the monoclonal antibody was a new human genetically engineered antibody against ZIKV, which laid the foundation for the early diagnosis of ZIKV, and obtain a specific monoclonal antibody to ZIKV for human treatment of ZIKV infection.

Influencia de variables sociodemográficas en el rendimiento de la Frontal Assessment Battery en adultos mayores Argentinos / Influence of sociodemographic variables on the performance of the Frontal Assessment Battery in Argentine older adults

La Frontal Assesment Battery (FAB) es una herramienta de screening de valoración rápida de las funciones ejecutivas. Estudios previos evidencian que variables sociodemográficas como la edad, el sexo y el nivel educativo podrían influir en el rendimiento de la FAB. El objetivo del estudio es analizar, en adultos mayores argentinos provenientes de la población general y sin deterioro cognitivo (evaluados según el Minimental, pruebas de fluidez verbal semántica y fonológica y el Test del Reloj) si existe diferencia en la FAB a partir de dichas variables. Metodología. Se analizó el rendimiento de 145 adultos mayores provenientes de la población general y sin deterioro cognitivo entre 60 y 90 años. Se realizaron estudios de diferencias de medias (t de Student y ANOVA), análisis univariante de la varianza, correlaciones (r de Pearson) y regresiones lineales simples para analizar la influencia de la edad, el sexo, el nivel educativo y el rendimiento cognitivo en la FAB. Resultados. Se observaron diferencias estadísticamente significativas en el puntaje total de la FAB en función del nivel educativo, pero no en función de la edad, ni el sexo. Asimismo, se encontró que el puntaje de la FAB se correlacionó con otras pruebas de screening cognitivo, como el Minimental, el Test del Reloj y fluidez verbal (fonológica y semántica). Conclusiones. El presente estudio presenta resultados que señalan la importancia de considerar el nivel educativo en la valoración de la FAB

the Frontal Assesment Battery (FAB) is a screening tool for a quick assessment of executive functions. Previous studies show that sociodemographic variables such as age, sex and educational level may influence the performance of the FAB. The objective of this study is to analyze, in elderly Argentines from the general population and without cognitive impairment (assessed according to the Minimental, semantic and phonological verbal fluency tests and the Clock Test) if there are differences in the FAB explained by these variables. Methodology. The performance of 145 elderly adults from the general population without cognitive impairment between 60 and 90 years was analyzed. Studies of mean differences (Student's t-test and ANOVA), univariate analysis of variance, correlations (Pearson's r) and simple linear regressions were performed to analyze the influence of age, sex, educational level and general cognitive performance in the FAB. Results. Statistically significant differences were observed in the total score of the FAB according to the educational level, but not according to age or sex. It was also found that the FAB score correlated with other cognitive screening tests, such as the Minimental, the Clock Test and verbal fluencies (phonological and semantic). Conclusions. The present study presents results that indicate the importance of considering the educational level in the assessment of the FAB

Review for Retrospective Exposure Assessment Methods Used in Epidemiologic Cancer Risk Studies of Semiconductor Workers: Limitations and Recommendations

This article aims to provide a systematic review of the exposure assessment methods used to assign wafer fabrication (fab) workers in epidemiologic cohort studies of mortality from all causes and various cancers. Epidemiologic and exposure–assessment studies of silicon wafer fab operations in the semiconductor industry were collected through an extensive literature review of articles reported until 2017. The studies found various outcomes possibly linked to fab operations, but a clear association with the chemicals in the process was not found, possibly because of exposure assessment methodology. No study used a tiered assessment approach to identify similar exposure groups that incorporated manufacturing era, facility, fab environment, operation, job and level of exposure to individual hazardous agents. Further epidemiologic studies of fab workers are warranted with more refined exposure assessment methods incorporating both operation and job titleand hazardous agents to examine the associations with cancer risk or mortality.

Quality of horse F(ab')2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab′)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. Results Horse F(ab′)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. Conclusions Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab′)2 immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab′)2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.(AU)

Preparation and identification human anti-Siglec-9 Fab fragment antibody / 中国免疫学杂志

Objective:The prokaryotic expression vector of human anti-Siglec-9 Fab fragment antibody was constructed and purified,while was identified. Methods:The variable and conserved regions of heavy chain and light chain were obtained by polymerase chain reaction respectively(PCR),which was combined by overlap extension PCR and was digested with restriction enzyme,and then it was transformed into Escherichia coli BL21 and was purified by His-trap Lambda Fab column and AKTA system. SDS-PAGE,ELISA and Western blot were used for the identification of human anti-Siglec-9 Fab fragment antibody. The effect of human anti-Siglec-9 Fab fragment antibody on regulating the mRNA expression of TNF-α,IL-1,IL-6,IL-8 was detected by real-time PCR. Results:Successfully obtained the chains of heavy and light, while constructed an activation human anti-Siglec-9 Fab fragment antibody which could specifically bind to Siglec-9 protein. The human anti-Siglec-9 Fab fragment antibody could specifically bind to Siglec-9 was confirmed by SDS-PAGE,ELISA and Western blot. The human anti-Siglec-9 Fab fragment antibody inhibited the mRNA expression of TNF-α,IL-1, IL-6,IL-8. Conclusion:Successful prokaryotic expression,purification,character analysis,and suppressed the mRNA expression of in-flammatory cytokines with the human anti-Siglec-9 Fab fragment antibody and lay the biology foundation for the further study.

Executive dysfunction in patients with idiopathic epilepsy

Background: Idiopathic epilepsy is defined as disorder in which there is no underlying cause or structural pathology other than a possible hereditary predisposition for generating seizures which can be generalized or focal in nature. Objective: To study the occurrence of executive dysfunction in patients with idiopathic epilepsy and its association with age, gender, seizure type, duration of epilepsy, age at seizure onset, antiepileptic drug therapy and seizure control &the association of the interictal EEG pattern. Materials and methods: 100 cases of epileptic patients with normal CT scan brain / MRI brain were studied. Frontal Assessment Battery &The Executive Interview (EXIT) were used to assess the executive functions. Results: The FAB score were normal in 46% and abnormal in 54% of the cases. Executive dysfunction as per the FAB score was mild in 32% and moderately severe in 22% of the cases. Impairment in Executive function as per EXIT score was mild in 84% and moderate in 16 % of the cases. Executive Dysfunction was more in cases with either primary or secondarily generalized seizures, cases with a higher seizure frequency, longer duration of epilepsy, uncontrolled epilepsy and seen in 17.5% with normal and 10% cases with an abnormal EEG. Conclusion: Our study found a significant proportion of patients with idiopathic epilepsy have Executive Dysfunction, which adds to the seizure burden by reducing the capacity of an individual to successfully engage in self-care, social, academic and occupational pursuits.

Quality of horse F(ab)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...

Selection and identification of human monoantibody TRAb Fab fragment / 天津医药

Objective To select and express a human thyrotrophin receptor antibody (TRAb) Fab fragment from phage antibody library constructed with phage display technology. Methods With immobilized antigen, the reconstructed humanized TRAb Fab library was enriched by five rounds panning (adsorption-elution-amplification). The TSAb Fab and TBAb Fab fragment were selected by coated fusion proteins of hTSHRn and hTSHRc. The positive clones were identified and selected by Phage-ELISA. TRAb positive clones were identified by PCR and double restriction enzyme digestion. The soluble TRAb (TSAb, TBAb) Fab fragments were expressed. TRAb (TSAb, TBAb) Fab fragments were identified by Western blotting assay. The DNA fragment was sequenced from the positive clones. Results Following five rounds of biopanning, TRAb (TSAb,TBAb) Fab phage antibody was screened. The enrichment effect reached to 77 times and 94 times. The soluble TRAb (TSAb,TBAb) Fab antibodies were expressed from positive clones and identified by phage ELISA. Western blotting analysis showed that the phage displaying Fab had significant binding activity with antigens. These sequence analysis showed that all of the heavy chain Fd gene and light chain gene were derived from human immunoglobulin variable region. The light chain variable region of the monoclonal 48 was homologous to the immunoglobulin light chain Vλ homology of 94.4%, and the heavy chain variable region of the monoclonal 48 was homologous to the immunoglobulin heavy Fd chain VH4 homology of 88.9%. The light chain variable region of the monoclonal 56 was homologous to the immunoglobulin light chain Vλ homology of 95.6%, and the heavy chain variable region of the monoclonal 56 was homologous to the immunoglobulin heavy Fd chain VH3 homology of 84.6%. Conclusion We have successfully selected TRAb (TSAb, TBAb) Fab fragment from a human phage display immune antibody library.

Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

PURPOSE: High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. MATERIALS AND METHODS: Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1a(tm1Knt) Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. RESULTS: NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. CONCLUSION: Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.

Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

PURPOSE: High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. MATERIALS AND METHODS: Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1a(tm1Knt) Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. RESULTS: NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. CONCLUSION: Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.

A respective analysis on 462 cases of leukemia typing / 国际检验医学杂志

Objective To study the relationship between FAB‐classifying standard and immunophenotype of leukemia in clinical diagnostic and treatment .Methods There were 462 cases of leukemia according to FAB‐classifying standard and 34 cases by immu‐nophenotype in our hospital from 2011 to 2013 .The results of FAB‐classifying standard and immunophenotype were statisticed and compared .Results Among the 462 cases ,there were 171 acute myeloid leukemia (AML) ,50 acute lymphoblastic leukemia (ALL) , 76 chronic leukemia ,3 rare types ,56 unclear types and 27 mismatched according the FAB‐classifying standard .The diagnostic rate was 82 .03% .34 cases of immunophenotype included 29 single phenotype ,4 double phenotypes and 1 unknown .The diagnosis rate was 97 .06% .CD13 and CD33 have high positive express in AML ;CD34 and HLA‐DR do not express in M3 ;CD7 which was known as a lymphatic antigen also was founded in AML .Conclusion The combing use of FAB‐classifying standard and immunophenotype can improve diagnostic rate ,and immunophenotype has an important role in differentiating different subtypes of leukemia .

Site-specific PEGylation of lidamycin and its antitumor activity

In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (M w 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs.

Targeting distribution of anti-CD19(Fab)-LDM:a new anti-lymphoma drug / 中国药理学通报

Aim To study targeting capability of anti-CD19 (Fab)-LDMto CD19 +B lymphoma cells in vi-vo and in vitro.Methods Flow cytometry was em-ployed to determine the affinity of Cy5 labeled anti-CD19 (Fab)-LDP to human lymphoma Raji cells.And the optical imaging system was used to analyze the dis-tribution of Cy5-anti-CD19 (Fab )-LDP in lymphoma-transplanted xenograft nude mice in vivo.Results The results of flow cytometry demonstrated that Cy5-an-ti-CD19(Fab)-LDP had remarkable affinity with lym-phoma Raji cells;Raji lymphoma xenograft model was established successfully in nude mice and in vivo fluo-rescence imaging analysis indicated that the antibody-drug conjugates could specially be localized in the tar-get tumor.Conclusion The experiments in vivo and vitro confirm that anti-CD19 (Fab)-LDP has remarka-ble affinity to targeting CD19 +lymphoma cells,and the antibody drugs anti-CD19 (Fab )-LDP have the probability to be new drugs for the treatment of malig-nant lymphoma.

A Case of Severe Digoxin Intoxication Treated with Digoxin Immune Fab

Digoxin is commonly used in treatment of various heart conditions, such as atrial fibrillation, atrial flutter, and sometimes heart failure. The therapeutic range of digoxin is narrow. Therefore, Digoxin toxicity is common. However, severe digoxin intoxication is uncommon. Many cases involving application of Digoxin immune Fab for digoxin intoxication in other countries have been reported. However, no cases have been reported in Korea. We reported on one case, a 34-year-old male with acute and severe digoxin intoxication who was treated with digibind(R). His chief complaint was gastrointestinal symptoms, including nausea and vomiting. Electrocardiography (ECG) showed third degree atrioventricular (AV) block. After an infusion of digibind(R), third degree AV block rhythm was changed to first degree AV block. Nowadays, we can obtain a digoxin antidote from the national poisoning information center. Therefore, we should actively consider application of Digoxin immune Fab in patients with severe digoxin intoxication.