ABSTRACT
@#Objective To explore the factors affecting the stability of high concentration variable domain of heavy-chain antibody-Fc(VHH-Fc) fusion protein.Methods Three groups of forced degradation experiments,shaking,light and 40℃ high temperature were set up.Differential scanning fluorimetry,dynamic light scattering(DLS) and ultra performance liquid chromatography-mass spectrometry(UPLC-MS) were used to detect the effects of the three forced degradation conditions on the conformational stability,colloidal stability,average hydrodynamic diameter and post-translational modifications of high concentration VHH-Fc fusion protein.Results Under the light condition,the onset temperature of unfolding(T_(onset)),melting temperature(T_m) and aggregation onset temperature(T_(agg)) of high concentration VHH-Fc fusion protein decreased the most,and the oxidation ratio of Met160 and Met266 increased significantly.Under the condition of shaking,the variation of the diffusion interaction parameter(k_D) and the average hydrodynamic diameter was the largest.Conclusion Light can significantly reduce the conformational stability of high concentration VHH-Fc fusion protein and induce methionine oxidation.Shaking has the most significant effect on its colloidal stability and promotes aggregation.
ABSTRACT
Los rotavirus del grupo A (RVA) constituyen la principal causa de diarrea grave y mortalidad infantil. La porción variable de los anticuerpos de cadena pesada derivados de camélidos presentan una amplia capacidad de unión antigénica (reconocen sitios antigénicos no accesibles a los anticuerpos tradicionales, con elevada afinidad) tienen bajos costos de producción y resultan ideales para las terapias orales. A la fecha, se desarrollaron 2 pares de nanoanticuerpos VHH contra RVA: ARP1-ARP3 y 2KD1-3B2. En este trabajo, exploramos el potencial de ambos grupos de nanoanticuerpos como estrategias de prevención complementarias a la vacunación y como una opción de tratamiento frente a la diarrea asociada a RVA en poblaciones de riesgo. Ambos pares de nanoanticuerpos fueron expresados en diferentes sistemas de producción y mostraron amplia capacidad neutralizante contra diversas cepas de RVA in vitro. También fueron usados en el modelo de ratón lactante, en el que evidenciaron distintos grados de éxito en la prevención o el tratamiento de la diarrea inducida por RVA. Es interesante destacar que la mitigación de los síntomas también se logró con ARP1 liofilizado y conservado, por lo que podría ser utilizado en áreas donde es difícil mantener la cadena de frío. Asimismo, 3B2 fue testeado en una prueba preclínica utilizando como modelo al cerdo gnotobiótico, al cual confirió completa protección contra la diarrea inducida por RVA. ARP1 fue usado en la primera prueba clínica de nanoanticuerpos VHH contra RVA, donde redujo significativamente las deposiciones en pacientes pediátricos con diarrea positivos para RVA, sin evidenciar ninguna reacción adversa
Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects
Subject(s)
Rotavirus/drug effects , Diarrhea/prevention & control , Single-Domain Antibodies/therapeutic use , Antibodies/therapeutic use , Primary Prevention/trends , Child Mortality , Single-Domain Antibodies/administration & dosageABSTRACT
Anticorpos, agentes empregados no desenvolvimento de pesquisas biomédicas, no diagnóstico e na terapêutica, possuem elevada capacidade de interação aos mais variados ligantes, Estruturalmente são heterotetrameros constituídos por duas cadeias leves e duas cadeias pesadas com massa molecular de aproximadamente 150 kDa, Visando melhorar as características farmacocinéticas e minimizar possíveis reações adversas desencadeadas por imunoglobulinas de origem não humana, a engenharia molecular de anticorpos vem obtendo fragmentos de anticorpos como porções Fab, F(ab?)2, scFv e Fv, Em adição aos anticorpos convencionais, camelídeos produzem imunoglobulinas funcionais desprovidas de cadeia leve, onde o domínio variável da cadeia pesada, denominado VHH ou nanocorpo, é responsável pelo reconhecimento antigênico, Apresentando características adequadas ao desenvolvimento de fármacos com alta capacidade de neutralização, fragmentos VHHs vêm sendo propostos para uso em imunoterapia passiva ou em drug-delivery, No diagnóstico esses fragmentos podem ser aplicados na construção de biosensores ou na imagiologia, atuando na detecção de células cancerígenas, no monitoramento de tumores ou em alterações celulares...
Antibodies, agents employed for the development of biomedical research, diagnostic and therapeutic, have high ability to interact with different ligands. Structurally are heterotetramers constituted by two light and two heavy chains, with molecular weight of approximately 150 kDa. Aiming to improve the pharmacokinetic properties and minimize possible adverse reactions triggered by immunoglobulins of non-human origin, the molecular engineering of antibodies has been obtaining fragments of antibodies, such as Fab, F(ab?)2, Fv and scFv. In addition to the conventional antibodies, camelids produce functional immunoglobulins devoid of light chain, in which the variable domain, named VHH or nanocorpo, is able to recognize the antigen. With appropriate characteristics for the development of drugs with high neutralizing capacity, VHH fragments have been proposed for use in passive immunotherapy or drug-delivery. To the diagnosis, these fragments can be used to construct biosensors, in the imagiology , acting in the detection of cancer cells, tumor monitoring or cell changes...